Mantoniella Squamata Research Articles

Robust optogenetic inhibition with red-light-sensitive anion-conducting channelrhodopsins

Channelrhodopsins (ChRs) are light-gated ion channels widely used to optically activate or silence selected electrogenic cells, such as individual brain neurons. Here, we describe identifying and characterizing a set of anion-conducting ChRs (ACRs) from diverse taxa and representing various branches of the ChR phylogenetic tree. The Mantoniella squamata ACR (MsACR1) showed high sensitivity to yellow-green light (λmax at 555 nm) and was further engineered for optogenetic applications. A single amino-acid substitution that mimicked red-light-sensitive rhodopsins like Chrimson shifted the photosensitivity 20 nm toward red light and accelerated photocurrent kinetics. Hence, it was named red and accelerated ACR, raACR. Both wild-type and mutant are capable optical silencers at low light intensities in mouse neurons in vitro and in vivo, while raACR offers a higher temporal resolution.

Robust optogenetic inhibition with red-light-sensitive anion-conducting channelrhodopsins.

Channelrhodopsins (ChRs) are light-gated ion channels widely used to optically activate or silence selected electrogenic cells, such as individual brain neurons. Here, we describe identifying and characterizing a set of anion-conducting ChRs (ACRs) from diverse taxa and representing various branches of the ChR phylogenetic tree. The Mantoniella squamata ACR (MsACR1) showed high sensitivity to yellow-green light (λmax at 555 nm) and was further engineered for optogenetic applications. A single amino-acid substitution that mimicked red-light-sensitive rhodopsins like Chrimson shifted the photosensitivity 20 nm toward red light and accelerated photocurrent kinetics. Hence, it was named red and accelerated ACR, raACR. Both wild-type and mutant are capable optical silencers at low light intensities in mouse neurons in vitro and in vivo, while raACR offers a higher temporal resolution.

Influence of thylakoid membrane lipids on the structure of aggregated light-harvesting complexes of the diatom Thalassiosira pseudonana and the green alga Mantoniella squamata.

The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light-harvesting complexes (LHCs). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana-stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the fucoxanthin chlorophyll protein (FCP) complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X-100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macroaggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid-induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana-stroma differentiation.

The investigation of violaxanthin de-epoxidation in the primitive green alga Mantoniella squamata (Prasinophyceae) indicates mechanistic differences in xanthophyll conversion to higher plants

Schaller S., Latowski D., JemioŁa-Rzemińska M., Quaas T., Wilhelm C., StrzaŁka K. and Goss R. 2012. The investigation of violaxanthin de-epoxidation in the primitive green alga Mantoniella squamata (Prasinophyceae) indicates mechanistic differences in xanthophyll conversion to higher plants. Phycologia 51: 359–370. DOI: 10.2116/11-127.1The violaxanthin (Vx) de-epoxidation of the green alga Mantoniella squamata (Prasinophyceae) was investigated in thylakoid membranes and in enzyme assays with the isolated Vx de-epoxidase (VDE), where pure Vx or purified light-harvesting complexes of M. squamata (MLHC) or spinach (LHCII) were used. The detailed analysis of the VDE amino acid sequences of prasinophycean algae and higher plants suggests that structural differences of the M. squamata VDE are responsible for the slow conversion of Ax to Zx that is typical for this alga. The present experiments further demonstrate that the availability of the substrate Vx has a strong impact on the overall de-epoxidation reaction and the ratio of the second de-epoxidation rate (de-epoxidation from Ax to Zx) to the first de-epoxidation rate (conversion from Vx to Ax). High concentrations of available Vx, as found in enzyme assays with pure Vx, lead to saturation of the VDE and a strong competition with the intermediate reaction product Ax, thus decreasing the ratio of the second de-epoxidation rate to the first de-epoxidation rate. In de-epoxidation assays with isolated LHCs, where the availability of Vx is limited by the detachment of Vx from the protein, an increased ratio of the second de-epoxidation rate to the first de-epoxidation rate is observed. The present study also shows that the interaction between the isolated LHC and the VDE can influence the ratio of the two de-epoxidation rates. Mantoniella squamata VDE, which exhibits a very low ratio of the second de-epoxidation rate to the first de-epoxidation rate in thylakoids or in enzyme assays with the MLHC, shows a high ratio when it is incubated with the LHCII of spinach.

Metabolic Engineering of ω3-Very Long Chain Polyunsaturated Fatty Acid Production by an Exclusively Acyl-CoA-dependent Pathway

omega3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Delta6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds.

Time-resolution of the Antheraxanthin- and ΔpH-dependent Chlorophyll a Fluorescence Components Associated with Photosystem II Energy Dissipation in Mantoniella squamata¶

The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (ΔpH), was examined in vivo.Mantoniella is distinguished from other photosynthetic organisms by two main features namely (1) a unique light-harvesting complex that serves both photosystems I (PSI) and II (PSII); and (2) a violaxanthin (V) cycle that undergoes only one de-epoxidation step in excess light to accumulate the monoepoxide antheraxanthin (A) as opposed to the epoxide-free zeaxanthin (Z). The cells were treated first with high light to induce the ΔpH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the ΔpH and nigericin-sensitive fluorescence level in the dark. De-epoxidation was controlled with subsaturating concentrations of dithiothreitol (DTT) and was 5–10 times more sensitive to DTT than higher plant thylakoids. The PSII energy dissipation involved two steps: (1) the pH activation of the xanthophyll binding site that was associated with a narrowing and slight attenuation of the main 2 ns (ns = 10−9 s) fluorescence lifetime distribution; and (2) the concentration-dependent binding of A to the activated binding site yielding a second distribution centered around 0.9 ns. Consistent with the model of Gilmore et al. (1998) (Biochemistry 37, 13 582–13 593), the fractional intensity of the 0.9 ns component depended almost entirely on the A concentration and correlated linearly with the decrease of the steady-state chlorophyll a fluorescence intensity.

Phylogenetic Position of the Green FlagellateMesostigma virideBased on α‐Tubulin and β‐Tubulin Gene Sequences

The green alga Mesostigma viride has attracted considerable recent attention as a model system useful for understanding the evolutionary origin of the Viridiplantae (all green algae plus embryophytes) or the Streptophyta (charophycean algae plus embryophytes). Mesostigma’s utility for such studies depends on its phylogenetic position, which has been controversial. Phylogenetic analyses based on organellar genes have given conflicting results; some support a hypothesis that Mesostigma represents the first branch of the Viridiplantae, whereas others support an alternative hypothesis that it occupies a basal position within Streptophyta. Nuclear‐encoded actin and small subunit ribosomal gene sequences also mark Mesostigma as a basal lineage in Streptophyta. We tested these two alternative hypotheses by analyses of nuclear‐encoded α‐ and β‐tubulin sequences. We obtained new α‐ and β‐tubulin gene sequences from M. viride as well as from the charophycean Coleochaete scutata and the green flagellates Pterosperma cristatum, Nephroselmis rotunda, and Mantoniella squamata, each representing a major clade within Prasinophyceae, in order to increase the taxon richness of the databases. We employed Bayesian and maximum likelihood procedures and paid particular attention to codon position effects on the phylogenetic analysis. On balance, our results support the hypothesis that M. viride is a basal streptophyte.

New Insights into the Nature and Phylogeny of Prasinophyte Antenna Proteins: Ostreococcus tauri, a Case Study

The basal position of the Mamiellales (Prasinophyceae) within the green lineage makes these unicellular organisms key to elucidating early stages in the evolution of chlorophyll a/b-binding light-harvesting complexes (LHCs). Here, we unveil the complete and unexpected diversity of Lhc proteins in Ostreococcus tauri, a member of the Mamiellales order, based on results from complete genome sequencing. Like Mantoniella squamata, O. tauri possesses a number of genes encoding an unusual prasinophyte-specific Lhc protein type herein designated "Lhcp". Biochemical characterization of the complexes revealed that these polypeptides, which bind chlorophylls a, b, and a chlorophyll c-like pigment (Mg-2,4-divinyl-phaeoporphyrin a5 monomethyl ester) as well as a number of unusual carotenoids, are likely predominant. They are retrieved to some extent in both reaction center I (RCI)- and RCII-enriched fractions, suggesting a possible association to both photosystems. However, in sharp contrast to previous reports on LHCs of M. squamata, O. tauri also possesses other LHC subpopulations, including LHCI proteins (encoded by five distinct Lhca genes) and the minor LHCII polypeptides, CP26 and CP29. Using an antibody against plant Lhca2, we unambiguously show that LHCI proteins are present not only in O. tauri, in which they are likely associated to RCI, but also in other Mamiellales, including M. squamata. With the exception of Lhcp genes, all the identified Lhc genes are present in single copy only. Overall, the discovery of LHCI proteins in these prasinophytes, combined with the lack of the major LHCII polypeptides found in higher plants or other green algae, supports the hypothesis that the latter proteins appeared subsequent to LHCI proteins. The major LHC of prasinophytes might have arisen prior to the LHCII of other chlorophyll a/b-containing organisms, possibly by divergence of a LHCI gene precursor. However, the discovery in O. tauri of CP26-like proteins, phylogenetically placed at the base of the major LHCII protein clades, yields new insight to the origin of these antenna proteins, which have evolved separately in higher plants and green algae. Its diverse but numerically limited suite of Lhc genes renders O. tauri an exceptional model system for future research on the evolution and function of LHC components.

Comparison by PAM Fluorometry of Photosynthetic Activity of Nine Marine Phytoplankton Grown Under Identical Conditions¶

ABSTRACTThe photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude‐modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII‐photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 μmol m−2 s−1) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 μmol m−2 s−1). These differences were interpreted to be the result of variations in the size of lightharvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.

Comparison of Photosynthetic Activity of Nine Different Marine Phytoplankton Grown Under Identical Conditions Using PAM Fluorometry

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude-modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII-photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 micromol m(-2) s(-1)) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 micromol m(-2) s(-1)). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.

Light regulation of carotenoid biosynthesis in the prasinophycean alga Mantoniella squamata.

We have studied carotenoid biosynthesis in the prasinophycean alga Mantoniella squamata, which contains a great variety of carotenoids belonging to both the alpha- and the beta-carotene (Car) biosyntheses pathways. This unusual carotenoid composition allowed us to address the problem of how biosynthesis on the alpha- and the beta-Car pathway is regulated in response to different light regimes. We found that illumination with 4 h of actinic high light (HL, 250 micromol m(-2) s(-1)), and culture growth in permanent light (PL, 60 micromol m(-2) s(-1)). induced the de novo synthesis of violaxanthin (Vx) cycle pigments belonging to the beta-Car pathway. Carotenoid synthesis on the alpha-Car biosynthesis pathway led to a strong accumulation of lutein (L) and dihydrolutein (DhL). Both the newly synthesised Vx cycle pigments and L/DhL can be regarded as intermediate pools of carotenoids that were converted to light-harvesting pigments in low light (LL) periods following the phases of HL illumination. This transition to the light-harvesting pigments included the conversion of Vx to neoxanthin (Nx) on the beta-Car pathway, and the transformation of L/DhL to prasinoxanthin (Px), the main light-harvesting pigment of M. squamata belonging to the alpha-carotenoids. Isolation of light-harvesting complexes from L-enriched M. squamata cells showed that both L and the Vx cycle pigments were loosely bound to the LHC apoprotein. This peripheral binding is in agreement with their proposed role as intermediate pigments in the biosynthesis of light-harvesting pigments, and should allow a smooth detachment from the protein in periods of LL when the synthesis of Nx and Px is stimulated. We conclude that carotenoid synthesis proceeds in general in an economical way: in both, the alpha- and the beta-Car pathway HL induces the formation of photoprotective pigments, which in LL periods following the HL illumination are not degraded, but on the contrary are converted to light-harvesting pigments.

Time-resolution of the antheraxanthin- and delta pH-dependent chlorophyll a fluorescence components associated with photosystem II energy dissipation in Mantoniella squamata.

The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (delta pH), was examined in vivo. Mantoniella is distinguished from other photosynthetic organisms by two main features namely (1) a unique light-harvesting complex that serves both photosystems I (PSI) and II (PSII); and (2) a violaxanthin (V) cycle that undergoes only one de-epoxidation step in excess light to accumulate the monoepoxide antheraxanthin (A) as opposed to the epoxide-free zeaxanthin (Z). The cells were treated first with high light to induce the delta pH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the delta pH and nigericin-sensitive fluorescence level in the dark. De-epoxidation was controlled with subsaturating concentrations of dithiothreitol (DTT) and was 5-10 times more sensitive to DTT than higher plant thylakoids. The PSII energy dissipation involved two steps: (1) the pH activation of the xanthophyll binding site that was associated with a narrowing and slight attenuation of the main 2 ns (ns = 10(-9) s) fluorescence lifetime distribution; and (2) the concentration-dependent binding of A to the activated binding site yielding a second distribution centered around 0.9 ns. Consistent with the model of Gilmore et al. (1998) (Biochemistry 37, 13,582-13,593), the fractional intensity of the 0.9 ns component depended almost entirely on the A concentration and correlated linearly with the decrease of the steady-state chlorophyll alpha fluorescence intensity.

Non-photochemical chlorophyll fluorescence quenching and structural rearrangements induced by low pH in intact cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae).

We have used circular dichroism (CD) spectroscopy and chlorophyll fluorescence induction measurements in order to examine low-pH-induced changes in the chiral macro-organization of the chromophores and in the efficiency of non-photochemical quenching of the chlorophyll a fluorescence (NPQ) in intact, dark-adapted cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae). We found that: (i) high proton concentrations enhanced the formation of chiral macrodomains of the complexes, i.e. the formation of large aggregates with long-range chiral order of pigment dipoles; this was largely independent of the low-pH-induced accumulation of de-epoxidized xanthophylls; (ii) lowering the pH led to NPQ; however, efficient energy dissipation, in the absence of excess light, could only be achieved if a substantial part of violaxanthin was converted to zeaxanthin and antheraxanthin in Chlorella and Mantoniella, respectively; (iii) the low-pH-induced changes in the chiral macro-organization of pigments were fully reversed by titrating the cells to neutral pH; (iv) at neutral pH, the presence of antheraxanthin or zeaxanthin did not bring about a sizeable NPQ. Hence, low-pH-induced NPQ in dark adapted algal cells appears to be associated both with the presence of de-epoxidized xanthophylls and structural changes in the chiral macrodomains. It is proposed that the macrodomains, by providing a suitable structure for long-distance migration of the excitation energy, in the presence of quenchers associated with de-epoxidized xanthophylls, facilitate significantly the dissipation of unused excitation energy.

Global fluorescence lifetime distribution analyses of PSII energy dissipation mechanisms at low physiological temperatures

This study documents the spectral and kinetic resolution of the main energy dissipation processes that influence the PSII photochemical yield in vivo at low physiological temperatures. The primary experimental approach involves global statistical analysis of both fluorescence lifetime distribution data and steady state fluorescence data from pulse-amplitude-modulation instruments and 77K excitation-emission spectral maps. The materials of interest include marine prasinophyte algae, eucalyptus seedlings and a selection of Arabidopsis mutants exhibiting varying xanthophyll and PsbS protein concentrations. Global statistical analysis of the time-resolved data was augmented with independent data including the steady-state intensity, pH-estimates, PsbS protein levels and xanthophyll concentrations. At low temperatures, Mantoniella squamata cells exhibited dark-sustained nigericin-sensitive and antheraxanthin-dependent energy dissipation components independent of PSII photoinhibitory damage. Arabidopsis leaves also exhibited low temperature induced and sustained energy dissipation components that correlated with the PsbS titre and xanthophyll levels. Further, the PsbS deficient mutants facilitated definitive resolution of fluorescence lifetime distribution changes associated with PSII photochemistry, xanthophyll dependent photoprotection and photoinhibition. Sun and frost exposed snow gum seedlings exhibited complex fluorescence spectral-kinetic characteristics including components that correlated with deepoxidized xanthophylls and other components obviously associated with protein synthesis and or structural reorganization of the PSII core inner antenna. It is concluded that fluorescence lifetime distribution analysis provides quantifiable information regarding separate populations of PSII as they are influenced by reaction center photochemistry and both photoprotective and photoinhibitory energy dissipation mechanisms.

Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the Q Y dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9° at 677 nm, 20° at 670 nm and 26° at 662 nm); the Q Y dipole of Chl c was found to be oriented at 29° with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the Q Y dipoles of Chl b to lie preferentially in the plane of the membrane (23° at 653 nm and 30° at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.

Enzymatic consumption of carbonyl sulfide (COS) by marine algae

We show that the marine algae Mantoniella squamata, Prymnesium parvum, and Amphidinium klebsii take up carbonyl sulfide (COS) from their surrounding medium. Inhibitor studies confirm that this COS uptake is catalyzed by the enzyme carbonic anhydrase, which was not detectable with conventional methods. As shown for M. squamata, the COS uptake can be dependent on the growth conditions. Furthermore, COS uptake shows a clear positive correlation with the COS concentration in the growth medium. The value of K1/2 for the COS uptake was estimated to be around 222 mol/m3. The COS consumption by the marine algae species investigated was estimated to be negligible compared to the photoproduction and hydrolysis of COS in seawater.

The xanthophyll cycle of Mantoniella squamata converts violaxanthin into antheraxanthin but not to zeaxanthin: consequences for the mechanism of enhanced non-photochemical energy dissipation

The prasinophycean alga Mantoniella squamata uses in vivo an incomplete violaxanthin cycle. Although the violaxanthin cycle in Mantoniella is capable of converting violaxanthin to zeaxanthin, in intact cells only antheraxanthin accumulates during periods of strong illumination. Antheraxanthin enhances non-photochemical quenching of chlorophyll fluorescence. Inhibition of antheraxanthin synthesis by the de-epoxidase inhibitor dithiothreitol abolishes increased thermal energy dissipation. Antheraxanthin-dependent non-photochemical quenching, like zeaxanthin-mediated non-photochemical quenching in higher plants, is uncoupler-sensitive. Mantoniella squamata cells cultivated at high light intensities contain higher amounts of violaxanthin than cells grown at low light. The increased violaxanthin-cycle pool size in high-light-grown Mantoniella cells is accompanied by higher de-epoxidation rates in the light and by a greater capacity to quench chlorophyll fluorescence non-photochemically. Antheraxanthin-dependent amplification of non-photochemical quenching is discussed in the light of recent models developed for zeaxanthin- and diatoxanthin-mediated enhanced heat dissipation.

Comparative growth rates and yields of ciliates and heterotrophic dinoflagellates

Growth rates, ingestion rates and grazer yields (grazer volume produced/prey volume consumed) were measured for six protozoan species (ciliates: Favella sp., Strombidinopsis acumina- tum, Uronema sp.; heterotrophic dinoflagellates: Amphidinium sp., Gymnodinium sp., Noctiluca scintillans) in laboratory batch culture experiments. Comparative growth data indicate that the prymnesiophyte Isochrysis galbana, the prasinophyte Mantoniella squamata, two cryptophyte species and several autotrophic dinoflagellate species were suitable foods for these grazers. When grown on optimized diets at 13°C, maximum ciliate growth rates (range 0.77-1.01 day1) uniformly exceeded maximum heterotrophic dinoflagellate growth rates (range 0.41-0.48 day1). A compilation of published data demonstrates that this growth rate difference persists across a range of ciliate and dino- flagellate taxa and cell sizes. Comparison of volume-specif ic ingestion rates and yields for the six species studied here showed that there was no single explanation for this growth rate disparity. Heterotrophic dinoflagellates exhibited both low ingestion rates and, in one case, low yields; ciliates were able to achieve higher growth rates via either higher ingestion rates or higher yields, depending on ciliate species. Volume yield increased over time throughout the exponential growth phase in nearly all experiments, suggesting variation in response to changing food concentrations or long-term acclimation to culture conditions. Higher maximum ciliate growth rates mean that these grazers have the potential to exercise tighter control over incipient blooms of their prey than do heterotrophic dinoflagellates. Despite their evident importance in planktonic food webs, we still know little of the basic biology of planktonic protozoa. Taxonomy, life cycles, successional patterns and effects of environment on ecological rates remain poorly described. Contrast this with the situation for the other major group of planktonic grazers: the metazooplankton. The large amount of biological information available for many of these species has enabled researchers to describe the implications for

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.

Three-dimensional crystallization of the light-harvesting complex from Mantoniella squamata (Prasinophyceae) requires an adequate purification procedure

We present a new purification procedure for the light-harvesting complex of Mantoniella squamata whereupon three-dimensional crystallization succeeded. Previous purification methods were based on density centrifugations as the only separating principle. We have extended this preparation procedure by applying anion-exchange and molecular-sieve chromatography techniques. Purity and stability of the complex were proved by denaturing and non-denaturing polyacrylamide-gel electrophoresis, and spectroscopic measurements. With respect to contaminating lipids the purified pigment-protein complex was examined by thin-layer chromatography and the aggregation and/or oligomeric states were investigated by gel filtration, fluorescence emission, and electron microscopy. Molecular-mass determination by gel filtration gave evidence for a trimeric organization of the purified native complex. Our purification procedure resulted in mixed protein-detergent micelles, small and homogeneous, the ideal material to start crystallization trials. We found involuntary aggregation to be easily detected by additional fluorescence emission at red-shifted wavelength (700 nm) as compared to the main emission at 680 nm. Electron-microscopic images proved this emission at 700 nm to be really due to aggregation, which could be prevented if sufficient detergent were present in the samples during the whole purification procedure. The ultimate proof that our efforts have been successful was the appearance of hexagonal crystals.