Purification and identification of chlorophyll c1 from the green alga Mantoniella squamata

The prasinophycean alga Mantoniella squamata contains besides chlorophyll a and b a third chlorophyll c-like pigment in its light-harvesting antenna. This third chlorophyll was purified by reverse phase and polyethylene chromatography in order to identify its chemical structure. The absorption and fluorescence spectra were measured not only from the doubly purified pigment, but also from its Mg-free derivates. The spectra were compared with those of authentic chlorophyll c and of Mg-2,4-desethyl-2,4-divinylpheoporphyrin a₅ monomethyl ester which was isolated from Rhodobacter capsulata. The results show that the pigment from Mantoniella agrees best with chlorophyll c₁. In order to clarify the spectral data, chlorophyll c₁ and c₂, the pigment from Mantoniella and Mg-2,4-desethyl-2,4-divinylpheoporphyrin a₅ monomethyl ester were resolved by polyethylene chromatography. The chromatographic analysis clearly shows that the pigment from Mantoniella comigrates with chlorophyll c₁ and not with the bacterial pigment or chlorophyll c₂. Mantoniella is the first organism which has been demonstrated to contain chlorophyll a, b and c.     

CHLOROPHYLL C PIGMENT PATTERNS IN 18 SPECIES (51 STRAINS) OF THE GENUS PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE)1

The pigment composition of 18 species (51 strains) of the pennate diatom Pseudo‐nitzschia was examined using HPLC. The carotenoid composition was typical for diatoms, with fucoxanthin (the major xanthophyll), diadinoxanthin, diatoxanthin, and β,β‐carotene. However, a diverse array of chl c pigments was observed in the studied strains. All Pseudo‐nitzschia strains contained chl a and chl c₂, traces of Mg‐2,4‐divinyl phaeoporphyrin a₅ monomethyl ester (MgDVP), and traces of a chl c₂–like pigment originally found in the haptophyte Pavlova gyrans. The distribution of chl c₁ and chl c₃ was variable among species (present in seven and 14 species, respectively). Based on chl c distribution, three major pigment types were defined: type 1 (chl c₁ + c₂, four species: P. australis, P. brasiliana, P. multiseries, and P. seriata), type 2 (chl c₁ + c₂ + c₃, three species: P. fraudulenta, P. multistriata, and P. pungens), and type 3 (chl c₂ + c₃, 11 species: P. arenysensis, P. calliantha, P. cuspidata, P. decipiens, P. delicatissima, P. galaxiae, P. mannii, P. pseudodelicatissima, P. subcurvata, P. cf. subpacifica, and a novel Pseudo‐nitzschia species). Type 1 and 2 species also shared the absence of a particular morphological character, the central nodule in the raphe, with the only exception of P. fraudulenta. The implications of such pigment diversity in chemotaxonomy, HAB monitoring, ecology, and phylogeny of Pseudo‐nitzschia species are discussed.     

OCCURRENCE OF PHYIWATED CHLOROPHYLL c IN ISOCHRYSIS GALBANA AND ISOCHRYSIS SP. (CLONE T-ISO) (PRYMNESIOPHYCEAE)1

The pigment composition of two clones of Isochrysis galbana Parke (CCMP 1323 and CCAP 927/1), and Isochrysis sp. (clone T-ISO) was analyzed by high-performance liquid chromatography using a polymeric octadecylsilica column. Fluorescent peaks with retention times higher than chlorophyll a were detected for all three clones. The corresponding pigments were isolated and characterized in terms of their visible absorbance and fluorescence spectra. The pigments were similar to phytol-substituted chlorophyll c, previously isolated from Emiliania huxleyi (Lohm.) Hay and Mohler and other species containing chlmophyll c₃. The presence of phytol-substituted chlorophyll c in I. galbana which lacked chlorophyll c₃, increases the diversity of chlorophyll patterns for the Haptophyta, which can be grouped, at present, into six different pigment types. This is the jrst observation of a haptophyte containing the apolar phytylated chlorophyll c-like pigment but lacking chlorophyll c₃.     

Light capture and pigment diversity in marine and freshwater cryptophytes

Phenotypic traits associated with light capture and phylogenetic relationships were characterized in 34 strains of diversely pigmented marine and freshwater cryptophytes. Nuclear SSU and partial LSU rDNA sequence data from 33 of these strains plus an additional 66 strains produced a concatenated rooted maximum likelihood tree that classified the strains into 7 distinct clades. Molecular and phenotypic data together support: (i) the reclassification of Cryptomonas irregularis NIES 698 to the genus Rhodomonas, (ii) revision of phycobiliprotein (PBP) diversity within the genus Hemiselmis to include cryptophyte phycocyanin (Cr‐PC) 569, (iii) the inclusion of previously unidentified strain CCMP 2293 into the genus Falcomonas, even though it contains cryptophyte phycoerythrin 545 (Cr‐PE 545), and (iv) the inclusion of previously unidentified strain CCMP 3175, which contains Cr‐PE 545, in a clade with PC‐containing Chroomonas species. A discriminant analysis‐based model of group membership correctly predicted 70.6% of the clades using three traits: PBP concentration · cell^?1, the wavelength of PBP maximal absorption, and habitat. Non‐PBP pigments (alloxanthin, chl‐a, chl‐c₂, α‐carotene) did not contribute significantly to group classification, indicating the potential plasticity of these pigments and the evolutionary conservation of the PBPs. Pigment data showed evidence of trade‐offs in investments in PBPs vs. chlorophylls (a +c₂).     

DETECTION OF CHLOROPHYLLS c1, c2 AND c3 IN PIGMENT EXTRACTS OF PRYMNESIUM PARWM (PRYMNESIOPHYCEAE)1

Three chlorophyll c-type pigments were separated by reversed-phase high Performance liquid chromatography and thin-layer chromatography from pigment extracts of the prymnesiophyte, Prymnesium parvum Carter. Based on spectral characteristics, retention times, and comparison with reference pigments isolated from the diatom Phaeodactylum tricornutum Bohlin, two of these pigments were identijied as chlorophyll c₁ and c₂. The other pigment was identified by its absorption spectrum and thin-layer chromatography retention times as the newly described chlorophyll c₃. However, in other prymnesiophytes so far examined, chlorophyll c₁ and chlorophyll c₃ were present with no chlorophyll c_l. The discovery of chlorophyll c₃ with chlorophyll c₁ and chlorophyll c₃ in Prymnesium parvum therefore represents the first report of this combination of pigments in prymnesiophytes.     

BIO-OPTICAL CHARACTERISTICS AND PHOTOADAPTIVE RESPONSES IN THE TOXIC AND BLOOM-FORMING DINOFLAGELLATES GYRODINIUM AUREOLUM,GYMNODINIUM GALATHEANUM,AND TWO STRAINS OF PROROCENTRUM MINIMUM1

Photoadaptive responses in the toxic and bloom-forming dinoflagellates Gyrodinium aureolum Hulbert, Gymnodinium galatheanum Braarud, and two strains of Prorocentrum minimum (Pavillard)Schiller were evaluated with respect to pigment composition, light-harvesting characteristics, carbon and nitrogen contents, and growth rates in shade- and light-adapted cells. The two former species were grown at scalar irradiances of 30 and 170 μmol · m ^?2 at a 12-h daylength at 20° C. The two strains of P. minimum were grown at 35 and 500 μmol. m^?2· s^?1 at a 2-h daylength at 20° C. For the first time, chlorophyll (chl) c₃, characteristic of several bloom-forming prymnesiophytes, was detected in G. aureolum and G. galatheanum. Photoadaptional status affected the pigment composition strongly, and the interpretation of the variation depended on whether the pigment composition was normalized per cell, carbon, or chl a. Species-specific and photoadaptional differences in chl a-specific absorption (°a_c, 400–700 nm) and chl a-normalized fluorescence excitation spectra of photosystem II fluorescence with or without addition of DCMU (°F and °F_DCMU 400–700 nm) were evident. Gyrodinium aureolum and G. galatheanum exhibited in vivo spectral characteristics similar to chl c₃-containing prymnesiophytes in accordance with their similar pigmentation. Prorocentrum minimum had in vivo absorption and fluorescence characteristics typical for peridinin-containing dinoflagellates. Species-specific differences in in vivo absorption were also observed as a function of package effect vs. growth irradiance. This effect could be explained by differences in intracellular pigment content, cell size/shape, and chloroplast morphology/numbers. Light- and shade-adapted cells of P. minimum contained 43 and 17% of photoprotective carotenoids (diadino + diatoxanthin) relative to chl a, respectively. The photoprotective function of these carotenoids was clearly observed as a reduction in °F and °F ^DCMU at 400–540 nm compared to °ac in light-adapted cells of P. minimum. Spectrally weighted light absorption (normalized to chl a and carbon, 400–700 nm) varied with species and growth conditions. The use of quantum-corrected and normalized fluorescence excitation spectra with or without DCMU-treated cells to estimate photosynthetically usable light is discussed. The usefulness of in vitro absorption and fluorescence excitation spectra for estimation of the degradation status of chl a and the ratio of chl a to total pigments is also discussed.     

SEPARATION OF CHLOROPHYLLS c1c2, AND c3 OF MARINE PHYTOPLANKTON BY REVERSED-PHASE-C18-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY1

We separated chlorophylls c₁ c₂, and c₃ of marine phytoplankton together with other pigments by a modification of the commonly applied reversed-phase-C18-high-performance liquid chromatography (RP-C18-HPLC) method. However, the chlorophyll c-like pigment 2,4, Mg-divinylpheoporphyrin as monomethyl ester, co-eluted with chlorophyll c₁. The method involves optimization of the mobile phase by using a very high ion strength solvent in combination with a high carbon loaded RP-C18 column. Fingerprints of the various taxonomic groups of algae can thus be developed in a single run, including separation of the carotenoids lutein and zeaxanthin.     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

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.     

BENTHIC AND PLANKTONIC ALGAL COMMUNITIES IN A HIGH ARCTIC LAKE: PIGMENT STRUCTURE AND CONTRASTING RESPONSES TO NUTRIENT ENRICHMENT1

We investigated the fine pigment structure and composition of phytoplankton and benthic cyanobacterial mats in Ward Hunt Lake at the northern limit of High Arctic Canada and the responses of these two communities to in situ nutrient enrichment. The HPLC analyses showed that more than 98% of the total pigment stocks occurred in the benthos. The phytoplankton contained Chrysophyceae, low concentrations of other protists and Cyanobacteria (notably picocyanobacteria), and the accessory pigments chl c₂, fucoxanthin, diadinoxanthin, violaxanthin, and zeaxanthin. The benthic community contained the accessory pigments chl b, chl c₂, and a set of carotenoids dominated by glycosidic xanthophylls, characteristic of filamentous cyanobacteria. The black surface layer of the mats was rich in the UV‐screening compounds scytonemin, red scytonemin‐like, and mycosporine‐like amino acids, and the blue‐green basal stratum contained high concentrations of light‐harvesting pigments. In a first bioassay of the benthic mats, there was no significant photosynthetic or growth response to inorganic carbon or full nutrient enrichment over 15 days. This bioassay was repeated with increased replication and HPLC analysis in a subsequent season, and the results confirmed the lack of significant response to added nutrients. In contrast, the phytoplankton in samples from the overlying water column responded strongly to enrichment, and chl a biomass increased by a factor of 19.2 over 2 weeks. These results underscore the divergent ecophysiology of benthic versus planktonic communities in extreme latitudes and show that cold lake ecosystems can be dominated by benthic phototrophs that are nutrient sufficient despite their ultraoligotrophic overlying waters.     

PHYTOPLANKTON PIGMENTS IN COASTAL LAKE MICHIGAN: DISTRIBUTIONS DURING THE SPRING ISOTHERMAL PERIOD AND RELATION WITH EPISODIC SEDIMENT RESUSPENSION1

Phytoplankton pigment distributions during the spring isothermal periods of 1998 and 1999 and their association with episodic sediment resuspension were characterized in coastal waters of southern Lake Michigan. Total and phylogenetic group chl a concentrations (derived using chemical taxonomy matrix factorization of diagnostic carotenoids) corresponded with assemblage and group biovolumes estimated from microscopic enumeration (P≤ 0.001). Diatoms and cryptophytes dominated assemblages and together typically comprised greater than 85% of relative chl a. Total chl a concentrations and both fucoxanthin·chl a ^{? 1} and alloxanthin·chl a ^{? 1} ratios were similar across depths (P> 0.05), indicating uniform distributions of and photophysiological states for assemblages and diatoms and cryptophytes, respectively, throughout the mixed water column. Total chl a concentrations were not always spatially uniform from near‐shore to offshore waters, with the greatest variability reflecting the influence of tributary inflows upon coastal assemblages. Sediment resuspension strongly influenced water column particle density and light climate; however, total and group chl a concentrations did not correspond with coefficients of K_d and suspended particulate matter concentrations (P> 0.05). The correspondence of both light attenuation and suspended particulate matter concentration with relative diatom chl a (P≤ 0.001) indicated an apparent association between sediment resuspension and diatoms. This, and the negative association (P≤ 0.0001) between relative diatom and cryptophyte chl a, corresponded with the spatial dominance of diatom and cryptophyte chl a in near‐shore and offshore waters, respectively. The presence of viable chl a and fucoxanthin within the surficial sediment layer, established this layer as a potential source of meroplanktonic diatoms for near‐shore assemblages.     

ISOLATION AND CHARACTERIZATION OF PARMALES (HETEROKONTA/HETEROKONTOPHYTA/STRAMENOPILES) FROM THE OYASHIO REGION,WESTERN NORTH PACIFIC1

A small siliceous species of marine phytoplankton, order Parmales (Heterokonta), was isolated and characterized for the first time with the aid of a fluorescent silicon tracer 2‐(4‐pyridyl)‐5‐([4‐(2‐dimethylaminoethylaminocarbamoyl)‐methoxy]phenyl)oxazole (PDMPO). This dye was easily detected by clear fluorescence in newly produced silica cell plates. Our isolate was surrounded by eight smooth plates without any ornamentation, suggesting a similarity to Triparma laevis B. C. Booth. TEM observation showed the typical ultrastructure of photosynthetic heterokontophytes; with two chloroplast endoplasmic reticulate membranes, a girdle lamella, three thylakoid lamellae, and mitochondrion with tubular cristae. Molecular phylogenetic analyses of SSU rDNA and rbcL genes showed that the parmalean alga was within the bolidophycean clade of autotrophic naked flagellates and a sister group of diatoms. HPLC analysis detected chl a, c₁ + c₂, and c₃; fucoxanthin; and diadinoxanthin as major photosynthetic pigments, and a composition that is shared with Bolidophyceae and diatoms. Together, these data indicate a close evolutionary relationship between Parmales, Bolidophyceae, and diatoms. The PDMPO‐staining procedure should accelerate isolation of other Parmales species, helping to establish their diversity and aiding quantitative study of their role in oceanic processes.     

RESPONSE OF THE PHOTOSYNTHETIC APPARATUS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) TO NITRATE,PHOSPHATE, OR IRON STARVATION1

The effects of nitrate, phosphate, and iron starvation and resupply on photosynthetic pigments, selected photosynthetic proteins, and photosystem II (PSII) photochemistry were examined in the diatom Phaeodactylum tricornutum Bohlin (CCMP 1327). Although cell chlorophyll a (chl a) content decreased in nutrient-starved cells, the ratios of light-harvesting accessory pigments (chl c and fucoxanthin) to chl a were unaffected by nutrient starvation. The chl a-specific light absorpition coefficient (a*) and the functional absorption cross-section of PSII (σ) increased during nutrient starvation, consistent with reduction of intracellular self-shading (i.e. a reduction of the “package effect”) as cells became chlorotic. The light-harvesting complex proteins remained a constant proportion of total cell protein during nutrient starvation, indicating that chlorosis mirrored a general reduction in cell protein content. The ratio of the xanthophylls cycle pigments diatoxanthin and diadinoxanthin to chl a increased during nutrient starvation. These pigments are thought to play a photo-protective role by increasing dissipation of excitation energy in the pigment bed upstream from the reaction centers. Despite the increase in diatoxanthin and diadinoxanthin, the efficiency of PSII photochemistry, as measured by the ration of variable to maximum fluorescence (F_v/F_m) of dark-adapted cells, declined markedly under nitrate and iron starvation and moderately under phosphate starvation. Parallel to changes in F_v/F_m were decreases in abundance of the reaction center protein D1 consistent with damage of PSII reaction centers in nutrient-starved cells. The relative abundance of the carboxylating enzyme, ribulose bisphosphate carboxylase/oxygenase (RUBISCO), decreased in response to nitrate and iron starvation but not phosphate starvation. Most marked was the decline in the abundance of the small subunit of RUBISCO in nitrate-starved cells. The changes in pigment content and fluorescence characteristics were typically reversed within 24 h of resupply of the limiting nutrient.     

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.     

TOXIN PROFILE,PIGMENT COMPOSITION,AND LARGE SUBUNIT RDNA PHYLOGENETIC ANALYSIS OF AN ALEXANDRIUM MINUTUM (DINOPHYCEAE) STRAIN ISOLATED FROM THE FLEET LAGOON,UNITED KINGDOM1

Paralytic shellfish toxins, pigment composition, and large subunit (LSU) rDNA sequence were analyzed for a clonal culture of Alexandrium minutum Halim isolated in 2000 from the coastal Fleet Lagoon, Dorset, United Kingdom. The HPLC pigment analysis revealed the presence of chl a, peridinin, and diadinoxanthin as major pigments and chl c₁+c₂ and c₃, diatoxanthin, and β‐carotene as minor components. The toxins responsible for paralytic shellfish poisoning were analyzed by HPLC with postcolumn derivatization and fluorescence detection. The paralytic shellfish poisoning toxin profile of the Fleet Lagoon strain of A. minutum in exponential growth phase was dominated by gonyautoxin‐3 up to 54%, whereas gonyautoxin‐2 made up 10% and saxitoxin (STX) 36%. The average toxicity of the culture was 3.8 pg STX Eq·cell^?1, and total toxin content varied from 5.6 fmol·cell^?1 on day 1 to a maximum of 16.8 fmol·cell^?1 during the early stationary phase. Sequence analysis of the LSU rDNA revealed the strain to be closely related to several European strains of A. minutum and one isolated from Australian waters, although most of these do not produce STX. The shallow Fleet Lagoon may provide a favorable environment for A. minutum to bloom, and the presence of highly potent saxitoxins in this strain indicates potential for future shellfish contamination.     

PHOTOSYNTHETIC PIGMENTS IN FIFTY-ONE SPECIES OF MARINE DIATOMS1

The photosynthetic pigments of 51 species (71 isolates) of tropical and sub-tropical diatoms from 13 out of 22 families were examined. These were the Thalassiosiraceae, Melosiraceae, Coscinodiscaceae, Rhizosoleniaceae, Biddulphiaceae, Chaetoceraceae, Lithodesmiaceae, Eupodiscaceae, Cymatosiraceae, Diatomaceae, Naviculaceae, Nitzschiaceae and Phaeodactylinaceae. Pigments were analyzed by cellulose and polyethylene thin-layer chromatography (TLC) and reverse-phase high-performance thin-layer chromatography (HPTLC). All species contained chlorophylls a and c₂ and the carotenoids carotene, fucoxanthin, diatoxanthin and diadinoxanthin. In addition, 14 species (20 isolates) contained one or more of four minor carotenoids, which were not identified further. One species, Thalassiothrix heteromorpha, contained small amounts of a 19′-butanoyloxyfucoxanthin-like pigment, in addition to fucoxanthin. Chlorophyll c₂ was present in all the diatoms tested and occurred together with chlorophyll c₁ in 88% of them. The presence of both chlorophylls c₁ and c₂ therefore can no longer be considered a universal characteristic of the diatom class. Where chlorophyll c₁ was absent or occurred in trace amounts only (8 species, 11 isolates), it was usually replaced by a new chlorophyll c pigment designated chlorophyll c₃, recently characterized from several prymnesiophytes and one chrysophyte. Exceptions were Nitzschia closterium (CS-114), which contained only chlorophyll c₂, and Nitzschia bilobata (CS-47), which contained all three chlorophylls (c₁, c₂ and c₃) in approximately equal amounts. Five species that contained chlorophylls c₁ and c₂ also contained chlorophyll c₃ in trace quantities Quantitative pigment analyses of the 71 isolates showed that chlorophyll concentrations ranged from 0.02 μg. 10⁶ cells^?1 in the smallest diatom, Extubocellulus spinifer, to 174.4 μg. 10⁶ cells^?1 in one of the largest diatoms, Coscinodiscus sp. under the standard growth conditions used. The mean molar ratio of chlorophyll a:c in the 72 isolates was 3.33, with a range of 1.65–7.25. The close similarity between diatom and prymnesiophyte pigmentation was confirmed. Each class has three patterns of pigmentation: viz species with chlorophylls c₁ and c₂ and‘true’fucoxanthin, species with chlorophylls c₃ and c₂ and‘true’fucoxanthin, and species with chlorophylls c₃ and c₂ and fucoxanthin derivatives.     

ISOLATION OF DI(HYDROXYMETHYL)DIHYDROXYPYRROLIDINE FROM THE CYANOBACTERIAL GENUS CYLINDROSPERMUM THAT EFFECTIVELY INHIBITS DIGESTIVE GLUCOSIDASES OF AQUATIC INSECTS AND CRUSTACEAN GRAZERS1

An effective glucosidase inhibitor was isolated from the cyanobacterial genus Cylindrospermum. Its chemical structure was determined by MS and NMR spectrometry to be di(hydroxymethyl)dihydroxypyrrolidine (DMDP; 2(R),5(R)‐bis‐(hydroxymethyl)‐3(R), 4(R)‐dihydroxypyrrolidine). Its identity was established by comparison with an authentic compound. All five species of Cylindrospermum investigated synthesized this compound but accumulated it to a different extent intracellularly. Particularly active producers were the axenic C. licheniforme (22 pmol·nmol chl a ^{? 1} 1 Received 5 March 2002. Accepted 2 October 2002. ) and a monoxenic unknown species of Cylindrospermum that contained the maximum amount (159 pmol·nmol chl a ^{? 1} 1 Received 5 March 2002. Accepted 2 October 2002. ). The major part of DMDP was found to be extracellular for all species investigated. The isolated compound inhibited digestive α‐ and β‐glucosidases isolated from crustacean zooplankton (IC₅₀ 19 and 49 nM, respectively). The bacterial 1‐deoxynojirimycin, which was used as a well‐studied reference glucosidase inhibitor, was less inhibitory (IC₅₀ 520 and 2190, respectively). Digestive enzymes of macrozoobenthos (chironomids, trichoptera, and ephemeroptera) were less sensitive to DMDP. The insect digestive β‐glucosidase was more effectively inhibited than the α‐glucosidase. Beside others, the ecological function of the glucosidase inhibitor may be the reduction of the digestibility of the cyanobacterium for grazers.     

Production of protochlorophyll,protopheophytin, and bacteriochlorophyll by the mutant A1a of Rhodopseudomonas capsulata

Summary Two carotenoid less mutant strains of Rhodopseudomonas capsulata were isolated. The strain A1a pho^- was not able to grow photosynthetically and to synthesize bacteriochlorophyll. However, this organism produced protochlorophyll (phytol ester of Mg-2-vinylpheoporphyrin a₅ monomethylester) and protopheophytin. Both pigments were excreted as a macromolecular complex. The intracellular membrane system was poorly developed. A revertant strain of A1a (pho⁺) was isolated which was able to grow anaerobically in the light as well as aerobically in the dark. The generation time under photosynthetic conditions amounted to 16 hrs whereas under aerobic conditions in the dark that was found to be 2.8 hrs. In addition to bacteriochlorophyll, which was found exclusively in the membrane fraction, protochlorophyll and protopheophytin were synthesized and excreted. A small amount of these pigments was also found intracellulary in the membrane fraction. The structure of the well developed intracytoplasmic membrane reticulum was described.Abbreviations EDTA Ethylenediamine tetraacetic acid-Na - R Rhodospirillum - Rps. Rhodopseudomonas     

A new spectrally distinct component in preparations of chlorophyll c from the micro-alga Emiliania huxleyi (Prymnesiophycease)

A new chlorophyll c pigment designated chlorophyll c₃ has been isolated from the coccolithophorid Emiliania huxleyi (Prymnesiophyceae) using reverse-phase high-performance thin-layer chromatography (HPTLC). Its spectral properties were compared with chlorophylls c₁ and c₂ from standard sources. Visible absorption maxima of the new pigment in diethyl ether were at 451, 585 and 625 nm with band ratios of 30.77, 3.79 and 1.00, respectively. Chlorophyll c₂ was present in approximately equal proportions to chlorophyll c₃, with maxima in diethyl ether at 447, 579 and 628 nm and band ratios of 12.26, 1.17 and 1.00, respectively. No chlorophyll c₁ was detected. The visible absorption spectra of the magnesium-free derivatives of both chlorophylls c₂ and c₃ from E. huxleyi in acetone were also recorded. The new chlorophyll c₃ pigment was chromatographically and spectrally distinct from a similar pigment, magnesium 2,4-divinylpheoporphyrin a₅ monomethyl ester, present in prasinophyte algae, with which it could have been confused.     

Three photosynthetic antenna porphyrins in a primitive green alga

Fluorescence excitation spectra (between 400–500 and 610–700 nm) for chlorophyll emission from particles and detergent extracts of the primitive green microalga, Mantoniella, were measured. The results showed that the prophyrin, magnesium 2,4-divinylpheoporphyrin a₅, which this alga accumulates in addition to Chl b, also can transfer excitation energy to Chl a, and therefore act as antenna for photosynthesis. Evidence was found that magnesium 2,4-divinylpheoporphyrin a₅ has a Soret band near 450 nm in vivo which further increases the light-harvesting capacity of these algae growing deep in the open ocean.