Non-hydrolysable macromolecular constituents from outer walls of Chlorella fusca and Nanochlorum eucaryotum

Abstract Many green microalgae possess a thin trilaminar outer wall (TLS) with a very high resistance to chemical degradation. TLS are known to play an important protective role in living cells. They are also selectively preserved during fossilization and thus provide a major contribution to the fossil organic matter of a number of sedimentary rocks. However, little information is available on TLS chemical structure. Examination of the TLS of Chlorella fusca (a lacustrine Chlorophycea) and of Nanochlorum eucaryotum (a recently discovered marine Chlorophycea) indicated that (i) they exhibit morphological features commonly observed in other green microalgae, (ii) their non-hydrolysable macrom…

Multiple Short Term Effects of UV-B Radiation on the Diatom Phaeodactylum Tricornutum

Increases in UV-B irradiance lead to many specific damaging effects upon the plants including damage of the thylakoid membrane, partial inhibition of PS II, decrease of chloroplast ATPase activity, loss of enzyme activities in the calvin cycle and alterations in pigment synthesis (1). Under natural conditions enhanced UV-B light is always accompanied by high intensities of photosynthetic active radiation (PAR). Damaging effects due to photoinhibitory PAR and UV-B light which lead to several oxygen radical species (2) could be reduced by photoprotection mechanisms. One of these protection mechanisms is the xanthophyll cycle. In higher plants and green algae violaxanthin is converted to zeaxa…

Incorporation of membrane proteins into lipid surface monolayers: Characterization by fluorescence and electron microscopies

The preparation of oriented protein samples is an attractive goal, e.g., to gain more detailed information from spectroscopic experiments. Our approach towards this aim was to prepare monolayers of phospholipids at the air-water interface and to incorporate the proteins into these ordered structures. Subsequently, we used the Langmuir-Boldgett (LB) transfer technique to obtain samples of oriented proteins on solid supports. — Incorporation was achieved by spreading the proteins from a detergent solution onto a prespread lipid monolayer on the water surface. We characterized successful incorporation by in situ fluoresence microscopy and by electron microscopy, and investigated the topology o…

Comparative analysis of the composition of two chlorophyll-b-containing light-harvesting complexes.

The major light-harvesting complexes from Mantoniella squamata (Prasinophyceae) and from Chlorella fusca (Chlorophyceae) were analyzed with respect to polypeptide composition and pigmentation. It was found that the polypeptides of Mantoniella are smaller than those of Chlorella and bind twice the amount of pigment. We assume that the amount of pigment per polypeptide is of ecological as well as of taxonomical importance.

Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata.

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.

The Molecular Analysis of the Light Adaptation Reactions in the Yellow‐green Alga Pleurochloris meiringensis (Xanthophyceae)

The xanthophycean alga Pleurochloris meiringensis was homocontinuously cultured under high light (16 W/m2) and low light (2 W/m2) conditions. In low light cells, the chlorophyll a content and the dry weight on per cell basis is increased, the maximal photosynthetic capacity per chlorophyll is decreased. The content of chlorophyll c, vaucheriaxanthin-ester and heteroxanthin is similar in both cultures, whereas the content of diadinoxanthin and s-carotene is twice as high in high light cultures. High light cells contain more photosystem I and cytochrome f per chlorophyll than low light cells, whereas the QB content is found to be unchanged. Therefore, the ratio reaction center II/reaction cen…

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 analysi…

Isolation of chlorophyll-protein complexes and quantification of electron transport components in Synura petersenii and Tribonema aequale

The chlorophyll-protein complexes of the yellow alga Synura petersenii (Chrysophyceae) and the yellow-green alga Tribonema aequale (Xanthophyceae) were studied. The sodiumdodecylsulfate/sodiumdesoxycholate solubilized photosynthetic membranes of these species yielded three distinct pigment-protein complexes and a non-proteinuous zone of free pigments, when subjected to SDS polyacrylamid gel electrophoresis. The slowest migrating protein was identical to complex I (CP I), the P-700 chlorophyll a-protein, which possessed 60 chlorophyll a molecules per reaction center in Tribonema and 108 in Synura. The zone of intermediate mobility contained chlorophyll a and carotenoids. The absorption spect…

Changes in yield ofin-vivo fluorescence of chlorophyll a as a tool for selective herbicide monitoring

Triazines and derivatives of phenylurea, which are often found in outdoor water samples, induce specific changes in the yield of thein-vivo chlorophyll α-fluorescence of PSII. These changes are correlated quantitatively with the concentration of the herbicides and can therefore be used to set-up a low-price monitor system. In order to detect selectively the herbicide-sensitive part of the fluorescence emission a pulse amplitude modulated fluorimeter was used. The bioassay system was optimised with respect to test organism, growing and measuring conditions. The relationship between fluorescence yield and herbicide concentrations were experimentally determined for the triazines atrazine and s…

The Supramolecular Structure of Photosystem II — Phycobilisome‐Complexes of Porphyridium cruentum

The structure and arrangement of phycobilisomes of the unicellular red alga Porphyridium cruentum is compared with the organization of the thylakoid freeze-fracture particles in order to determine the relationship between phycobilisomes and photosystem II. The hemi-ellipsoidal phycobilisomes, 20 nm thick, are predominantly organized into rows; their centre to centre periodicity is 30–40 nm, so that they are well separated by a gap of 10–20 nm. The phycobilisomes are cleaved by a central faint furrow, parallel to the long axis from top to base. The organization of the exoplasmic particles in rows is similar to the arrangement of the phycobilisomes so that a structural relationship between bo…

Fluorescence induction kinetics as a tool to detect a chlororespiratory activity in the prasinophycean alga, Mantoniella squamata

Abstract Pulse-modulated fluorescence measuring systems were used to study the fluorescence induction kinetics of the primitive prasinophycean alga, Mantoniella squamata . DCMU-treated cells show a very strong non-photochemical quenching which is insensitive to uncouplers and can not be attributed to state transitions or photoinhibition. In order to analyze the origin for this quenching, different inhibitors of the photosynthetic electron flow were applied. It was found that DCCD, an inhibitor of the ATP-synthase as well as of the cytochrome- c oxidase, enhances the fluorescence quenching, whereas antimycin A, which blocks the cyclic electron flow around PS I, stimulates it. The effect of b…

Die Lichtsammelkomplexe der verschiedenen Algenstämme Phylogenetische Vielfalt eukaryotischer Photosyntheseapparate

Pigment-pigment interactions and secondary structure of reconstituted algal chlorophyll a/b-binding light-harvesting complexes of Chlorella fusca with different pigment compositions and pigment-protein stoichiometries

Earlier we have shown by in vitro reconstitution experiments that the pigment composition of the chlorophyll alb-binding light-harvesting complex of the green alga Chlorella fusca could be altered in a relatively broad range (Meyer and Wilhelm 1993). In this study we used these reconstituted complexes of different pigment loading to analyze the excitonic interactions between the pigment molecules and the secondary structure by means of circular dichroism spectra in the visible and the far UV spectral regions, respectively. We found that, in contrast to the expectations, the pigment composition and pigment content hardly affected the circular dichroism spectra in the visible spectral region.…

Energy transfer and pigment composition in three chlorophyll b-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella fusca (Chlorophyceae) and Sinapis alba.

Light-harvesting Chl a/b protein complexes were isolated from the higher plant Sinapis alba, the green alga Chlorella fusca, and the prasinophycean alga Mantoniella squamata by mild gel electrophoresis. The energy transfer from chlorophyll b and the accessory xanthophyll was measured by means of fluoresence spectroscopy at 77 K. The pigment composition of the isolated antenna complexes was determined by high performance liquid chromatography in order to calculate the number of light absorbing molecules per chlorophyll a in the different light-harvesting complexes. These results were complemented by the quantitation of the pigments in total thylakoids as well as in the different electrophore…

Trastuzumab therapy vs tetracycline controlled ERBB2 downregulation: influence on tumour development in an ERBB2-dependent mouse tumour model

Trastuzumab (Herceptin) has improved therapy of breast cancer. Only patients overexpressing ERBB2 are treated with trastuzumab, whereas its use in tumours without ERBB2 expression is useless. This led to the concept that the subgroup of trastuzumab-sensitive tumours is ‘ERBB2-dependent', meaning that ERBB2 signalling is indispensable for growth of these tumours. We used a mouse model that allows anhydrotetracycline (ATc)-controlled downregulation of ERBB2 in tumour tissue. ERBB2 mRNA and protein expression were downregulated below detection limit leading to a macroscopically complete tumour remission within 14 days. Tumour remission was accompanied by a strong decrease in proliferation, a m…

Isolation and characterization of a photosystem I-associated antenna (LHC I) and a photosystem I—core complex from the chlorophyll c-containing alga Pleurochloris meiringensis (Xanthophyceae)

Abstract A photosystem (PS) I holocomplex was isolated from Pleurochloris meiringensis Vischer (Xanthophyceae) using sucrose density centrifugation. This complex exhibited a fluorescence emission maximum at 715 nm, which is in accordance with the long wavelength emission of whole cells. The complex was further dissociated into a core complex and a light-harvesting protein (LHC I). The core protein contains mainly Chl a and β-carotene, is 8.25 times enriched in P700 and has its main emission maximum at 715 nm. Therefore, the longest wavelength emission of P. meiringensis is due to the PS I core, which is in contrast to higher plants. The LHC I differs from LHC II with regard to its polypepti…

In vivo ANALYSIS OF SLOW CHLOROPHYLL FLUORESCENCE INDUCTION KINETICS IN ALGAE: PROGRESS, PROBLEMS AND PERSPECTIVES

Some Critical Remarks on the Suitability of the Concept of the Photosynthetic Unit in Photosynthesis Research and Phytoplankton Ecology

Based on the classical definition of the photosynthetic unit after Emerson and Arnold, new definitions are presented and their consistency subjected to critical reflection. Modern molecular approaches to determine the physiological condition or even the efficiency of the photosynthetic machinery via determination of the magnitude of the photosynthetic unit are discussed against the background of new data on the dynamics of the thylakoid membrane.

The energy distribution between the photosystems and light-induced changes in the stoichiometry of system I and II reaction centers in the chlorophyll b-containing alga Mantoniella squamata (Prasinophyceae).

The chlorophyll b-containing alga Mantoniella squamata was analyzed with respect to its capacity to balance the energy distribution from the light-harvesting antenna to photosystem I or photosystem II. It was shown, that this alga is unable to alter the absorption cross section of the two photosystems in terms of short-time regulations (state transitions). The energy absorbed by the LHC, which contains 60% of total photosynthetic pigments, is transferred to both photosystems without any preference. The stoichiometry of the two photosystems is found to be extremely unequal and variable during light adaptation. In high light, the molar ratio of P-680 per P-700 is found to be two, whereas unde…

Nanochlorum eucaryotum: a Very Reduced Coccoid Species of Marine Chlorophyceae

Nanochlorum eucaryotum was isolated from a sea water aquarium housing different sponge species, cucumarias, small crustaceans and annelids. This bright green marine alga differs from all other known coccoid species. Its most prominent features are its very small cell size (1.5 pm) and its reduced cellular organization. Its cell contains one nucleus, one chloroplast, one mitochondrium and small vacuoles. Sometimes a Golgi apparatus can be seen. No other subcellular features have been observed. The cell wall is thin and smooth and does not contain any material of high electron density; only dividing cells show a rougher surface. The cells split into two daughter cells. No sexual reproduction …

Ultrastructure and freeze-fracture studies of the thylakoids ofMantoniella squamata (Prasinophyceae)

The ultrastructure and the supramolecular organization of the thylakoids of the small green flagellate,Mantoniella squamata, were examined in thin sections and freeze-fracture preparations. The whole chloroplast is tightly packed with thylakoids, which show a pattern of meandering, branching and/or anastomosing membranes. In freeze-fracture preparations only two fracture-faces can be distinguished: the PF- and the EF-face. The PF-face has a much higher particle density than the EF-face (PF: 4086 particles/μm2; EF: 865 particles/μm2). The EF-face is not as uniform as the PF-face. The areas which are packed with particles probably correspond to closely appressed thylakoid regions or adhesive …

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using (31)P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L beta) to liquid-crystalline (L alpha) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This pha…

The light-harvesting system of Euglena gracilis during the cell cycle

The apoproteins of the light-harvesting chlorophyll-protein complexes LHCI and CP29 (apparent molecular weights of 27 kDa and 29 kDa, respectively) of Euglena gracilis were identified immunologically. Both complexes are present in the thylakoids of autotrophically cultured Euglena cells during the whole cell cycle. The relative amount of each apoprotein tends to increase towards the end of the cell cycle. The light-harvesting chlorophyll-protein complex of photosystem II, LHCII, of E. gracilis contains chlorophyll a, chlorophyll b, neoxanthin, diadinoxanthin and beta-carotene. Its chlorophyll a/b ratio is about 1.7 during the whole cell cycle. About 9 h after cell division the ratio of diad…

The molecular architecture of the thylakoid membrane from various classes of eukaryotic algae

There is convincing consensus that the photosynthetic apparatus is of prokaryotic origin. The wide variety of algal plastids is mostly assumed to be the result of different endocytological events. Chloroplasts surrounded by two membranes as in rhodophytes and chlorophytes were considered as the association of a prokaryotic symbiont and a eukaryotic host, whereas algae having a chloroplast surrounded with more than two membranes can be delineated from an endocytological event of two eukaryotes (see S. Gibbs in this volume). Since chlorophyll b was neither combined with chlorophyll c nor with phycobiliproteins it was proposed that all present day chloroplasts can be integrated in three lines.…

The Interaction of State Transitions and Chlororespiration in the Xanthophycean Alga Pleurochloris Meiringensis

Wavelength dependent State I-State II-transitions have been shown to exist in chlorophytes and red algae. Little is known about the regulation of energy distribution between the photosystems of chlorophyll c-containing plants. Previously it was shown that in the xanthophycean alga Pleurochloris meiringensis two states of energy distribution could be established [l]: In state “D” light is preferentially transferred to PS II, whereas in state “L” PS I is favoured. These state regulations strictly depend on the intensity and not on the wavelength of prei1lumination. In this paper we give new evidence that chlororespiration is involved in the mechanism of state “L”-state “D”-transitions.

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

Abstract 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 a 5 monomethyl ester which was isolated from Rhodobacter capsulata . The results show that the pigment from Mantoniella agrees best with chlorophyll c 1 . In order…

The importance of a highly active and DeltapH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae.

The present study focuses on the regulation of diatoxanthin (Dtx) epoxidation in the diadinoxanthin (Ddx) cycle containing algae Phaeodactylum tricornutum, Thalassiosira pseudonana, Cyclotella meneghiniana and Prymnesium parvum and its significance for the control of the photosystem II (PS II) antenna function. Our data show that Dtx epoxidase can exhibit extremely high activities when algal cells are transferred from high light (HL) to low light (LL). Under HL conditions, Dtx epoxidation is strongly inhibited by the light-driven proton gradient. Uncoupling of the cells during HL illumination restores the high epoxidation rates observed during LL. In Ddx cycle containing algae, non-photoche…

Role of hexagonal structure-forming lipids in diadinoxanthin and violaxanthin solubilization and de-epoxidation

In this study, we have examined the influence of different lipids on the solubility of the xanthophyll cycle pigments diadinoxanthin (Ddx) and violaxanthin (Vx) and on the efficiency of Ddx and Vx de-epoxidation by the enzymes Vx de-epoxidase (VDE) from wheat and Ddx de-epoxidase (DDE) from the diatom Cyclotella meneghiniana, respectively. Our results show that the lipids MGDG and PE are able to solubilize both xanthophyll cycle pigments in an aqueous medium. Substrate solubilization is essential for de-epoxidase activity, because in the absence of MGDG or PE Ddx and Vx are present in an aggregated form, with limited accessibility for DDE and VDE. Our results also show that the hexagonal st…

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H(II) phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H(II) phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the con…

The Variability of the Photosynthetic Unit in Chlorella Fusca

The concept of the photosynthetic unit (PSIT) was introduced by Gaffron and Wohl (1936) on the basis of Emerson and Arnold’s flash light experiments (1932). The PSU was defined as the minimum of chlorophyll molecules required for the evolution of one O2. Later on, the PSU was considered to be the complex of one reaction center and its appropriate antenna of light harvesting chlorophyll molecules. Assuming that there is an equal number of the PS I and PS II reaction centers the PSU was mostly expressed as Chl/P-700. However, recently it could be shown that the ratio of PS II/PS I is variable depending on the light conditions during growth (Kawamura et al., 1979; Myers, Graham, 1982). Further…

Ancient recruitment by chromists of green algal genes encoding enzymes for carotenoid biosynthesis.

Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-…

Changes in pigmentation of phytoplankton species during growth and stationary phase — consequences for reliability of pigment-based methods of biomass determination

In applied water ecology several methods for estimating the biomass or activity of phytoplankton depend on the proportion of accessory pigments (xanthophylls) to chlorophyll a. Therefore, changes in pigmentation during growth and stationary phase were investigated in four different species (Amphidinium klebsii, Euglena gracilis, Prymnesium parvum, Cryptomonas ovata) typical representatives of the major algal groups. The ratios of the different xanthophylls to chlorophyll a depended not only on the growth phase, but also on the species. InAmphidinium andEuglena, the ratio of xanthophylls to chlorophyll rises continuously during the growth phase and declined during the stationary phase. InPry…

Growth and Photosynthesis of Nanochlorum eucaryotum, a New and Extremely Small Eucaryotic Green Alga

Nanochlorum eucaryotum is a very small species of unicellular coccoid green algae (1.5 pm). The growth of Nanochlorum under different conditions of salinity, pH and light intensities was studied. Optimal growth rates were observed with normal sea water salinity and low light conditions at pH 7.0. The contents of chlorophylls, carotinoids, soluble proteins and the chlorophyll a to chlorophyll b ratio were measured. The light saturating curves of Nanochlorum cells grown under light intensities of 100 lx, 2000 lx and 10000 lx reveal a very narrow capacity of light adaptation. When cultured under higher light intensities, Nanochlorum was not able to reach high photosynthetic activities but unde…

THE LIGHT-HARVESTING SYSTEM OF THE UNICELLULAR ALGA Mantoniella squamata (PRASINOPHYCEAE): EVIDENCE FOR THE LACK OF A PHOTOSYSTEM I-SPECIFIC ANTENNA COMPLEX

The light-harvesting complexes (LHC) were isolated from the unicellular alga Mantoniella squamata (Prasinophyceae) by sucrose-density centrifugation. Beside the major LHC (II), a photosystem I complex was obtained that could be dissociated into a photosystem I core complex and an associated LHC I. In contrast to other chlorophyll b-containing antennae, both LHC II as well as LHC I were observed to be identical with respect to the following features: the molecular weights, the isoelectric points and the retention behavior on anion-exchange chromatography of the apoproteins, the pigment content and the absorption and fluorescence spectra

Reconstitution of Light-Harvesting Complexes from Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae)

Abstract Reconstitution experiments of light-harvesting complexes were performed with the green alga Chlorella fusca and the chlorophyll c-containing prasinophyte Mantoniella squamata using a modified method according to Plumley and Schmidt [Proc. N atl. Acad. Sei. U .S.A . 84, 146 -150 (1987)]. Changing the pigment supply quantitatively or qualitatively in the reconsti­tution mixture homologous and heterologous reconstitutes were obtained. In contrast to higher plants, light-harvesting polypeptides from green algae are able to bind the chlorophylls as well as the xanthophylls in different stoichiometries. Heterologous reconstitutes of M . squamata polypeptides give further evidence for a r…