Histidine Ligated Iron-Sulfur Peptides.

Iron-sulfur clusters are thought to be ancient cofactors that could have played a role in early protometabolic systems. Thus far, redox active, prebiotically plausible iron-sulfur clusters have always contained cysteine ligands to the cluster. However, extant iron-sulfur proteins can be found to exploit other modes of binding, including ligation by histidine residues, as seen with [2Fe-2S] Rieske and MitoNEET proteins. Here, we investigated the ability of cysteine- and histidine-containing peptides to coordinate a mononuclear Fe2+ center and a [2Fe-2S] cluster and compare their properties with purified iron-sulfur proteins. The iron-sulfur peptides were characterized by UV-vis, circular dichroism, and paramagnetic NMR spectroscopies and cyclic voltammetry. Small (≤6 amino acids) peptides can coordinate [2Fe-2S] clusters through a combination of cysteine and histidine residues with similar reduction potentials as their corresponding proteins. Such complexes may have been important for early cell-like systems.

Bioactive molecules from ciliates: Structure, activity, and applicative potential.

Ciliates are a rich source of molecules synthesized to socialize, compete ecologically, and interact with prey and predators. Their isolation from laboratory cultures is often straightforward, permitting the study of their mechanisms of action and their assessment for applied research. This review focuses on three classes of these bioactive molecules: (i) water-borne, cysteine-rich proteins that are used as signaling pheromones in self/nonself recognition phenomena; (ii) cell membrane-associated lipophilic terpenoids that are used in interspecies competitions for habitat colonization; (iii) cortical granule-associated molecules of various chemical nature that primarily serve offence/defense functions.

Insect cold-tolerance and lipidome: Membrane lipid composition of two chironomid species differently adapted to cold.

Cell-membrane fluidity is a fundamental parameter in cold resistance. It is regulated by a fine tuning of lipid composition, usually involving a great chemical diversity among head-groups, chain lengths, and degree of unsaturation. To give new insights on Alpine chironomid cold adaptation, we analysed the lipid membrane composition of Diamesa tonsa and Pseudodiamesa branickii, two species known to have different cold-tolerance, stronger in the former. Membrane lipid composition was analysed by NMR and HPLC-MS in larvae under natural (4 °C) and laboratory conditions (30 min at - 4 °C). In both species the major class of membrane lipids were phosphatidylethanolamine (PE), reaching 93% in D. tonsa and 80% in P. branickii, followed by a minor relative amount of phosphatidylcholine (PC). Phospholipids (PL) acyl chains were highly unsaturated given the presence of a relevant amount of polyunsaturated fatty acid (PUFA), among which a high proportion of ω-3 chains. This study demonstrated that these species have a similar lipidome (e.g. relevant amount of PUFA and predominance of PE), but with relevant differences on which to base different membrane fluidity: (i) a higher unsaturation index and chain length of both PE and PC and a higher ratio PE/PC ratio in D. tonsa than in P. branickii; (ii) the absence of modifications in the lipid composition in D. tonsa under sub-zero temperature. These differences might support the different cold-tolerance of the two species. In fact, we suggest that the high PE/PC ratio and the low sterols content (as in D. tonsa) could be involved in the formation of highly deformable membranes increasing their capacity to survive freezing. Interestingly, LC-MS analysis of D. tonsa lipidome revealed a new class of lipids that we named 'PpC', absent in P. branickii, that is worth investigating.

Complexing the Marine Sesquiterpene Euplotin C by Means of Cyclodextrin-Based Nanosponges: A Preliminary Investigation.

Euplotin C is a sesquiterpene of marine origin endowed with significant anti-microbial and anti-tumor properties. Despite the promising functional profile, its progress as a novel drug candidate has failed so far, due to its scarce solubility and poor stability in aqueous media, such as biological fluids. Therefore, overcoming these limits is an intriguing challenge for the scientific community. In this work, we synthesized ß-cyclodextrin-based nanosponges and investigated their use as colloidal carriers for stably complex euplotin C. Results obtained proved the ability of the carrier to include the natural compound, showing remarkable values of both loading efficiency and capacity. Moreover, it also allowed us to preserve the chemical structure of the loaded compound, which was recovered unaltered once extracted from the complex. Therefore, the use of ß-cyclodextrin-based nanosponges represents a viable option to vehiculate euplotin C, thus opening up its possible use as pharmacologically active compound.

Bacterial outer membrane vesicles engineered with lipidated antigens as a platform for Staphylococcus aureus vaccine.

Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 µg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.

Functional Study of Lipoxygenase-Mediated Resistance against Fusarium verticillioides and Aspergillus flavus Infection in Maize.

Mycotoxin contamination of maize kernels by fungal pathogens like Fusarium verticillioides and Aspergillus flavus is a chronic global challenge impacting food and feed security, health, and trade. Maize lipoxygenase genes (ZmLOXs) synthetize oxylipins that play defense roles and govern host-fungal interactions. The current study investigated the involvement of ZmLOXs in maize resistance against these two fungi. A considerable intraspecific genetic and transcript variability of the ZmLOX family was highlighted by in silico analysis comparing publicly available maize pan-genomes and pan-transcriptomes, respectively. Then, phenotyping and expression analysis of ZmLOX genes along with key genes involved in oxylipin biosynthesis were carried out in a maize mutant carrying a Mu transposon insertion in the ZmLOX4 gene (named UFMulox4) together with Tzi18, Mo17, and W22 inbred lines at 3- and 7-days post-inoculation with F. verticillioides and A. flavus. Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while UFMulox4 was highly susceptible to both pathogens with the most elevated mycotoxin content. F. verticillioides inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to A. flavus. Additionally, oxylipin analysis revealed prevalent linoleic (18:2) peroxidation by 9-LOXs, the accumulation of 10-oxo-11-phytoenoic acid (10-OPEA), and triglyceride peroxidation only in F. verticillioides inoculated kernels of resistant genotypes.

Mesorhizobium comanense sp. nov., isolated from groundwater.

Strain 3P27G6T was isolated from an artesian well connected to the thermal water basin of Comano Terme, Province of Trento, Italy. In phylogenetic analyses based on multilocus sequence analysis, strain 3P27G6T clustered together with Mesorhizobium australicum WSM2073T. Genome sequencing produced a 99.51 % complete genome, with a length of 7 363 057 bp and G+C content of 63.53 mol%, containing 6897 coding sequences, 55 tRNA and three rRNA. Average nucleotide identity analysis revealed that all distances calculated between strain 3P27G6T and the other Mesorhizobium genomes were below 0.9, indicating that strain 3P27G6T represents a new species. Therefore, we propose the name Mesorhizobium comanense sp. nov. with the type strain 3P27G6T (=DSM 110654T=CECT 30067T). Strain 3P27G6T is a Gram-negative, rod-shaped, aerobic bacterium. Growth condition, antibiotic susceptibility, metabolic and fatty acid-methyl esters profiles of the strain were determined. Only few nodulation and nitrogen fixation genes were found in the genome, suggesting that this strain may not be specialized in nodulation or in nitrogen fixation.

Improving the Phloroglucinolysis Protocol and Characterization of Sagrantino Wines Proanthocyanidins.

Proanthocyanidins are key metabolites that explain wine sensorial character (bitterness and astringency) and red wine color changes during aging. Therefore, a fast and accurate method to evaluate the degree of polymerization and the structural composition of the polymeric proanthocyanidins is a crucial analytical tool. Phloroglucinolysis is the most used method for this analysis but, unfortunately, the phloroglucinol adducts of the monomeric flavan-3-ols are not commercially available, making the results less accurate. The aim of this work was the isolation by semi-preparative high performance liquid chromatography (HPLC) of these non-commercial compounds and their use for the development of an accurate UHPLC-MS/MS protocol. The purity of each adduct was established via quantitative 1H-nuclear magnetic resonance (NMR) measurements with 3-trimethylsilyl-propionic-d4 acid sodium salt as the calibration standard. The developed method was applied to evaluate the proanthocyanidins profile of Sagrantino di Montefalco wines in comparison to other well-known tannic wines. Commercial, 6-8 years old Sagrantino wines were demonstrated to be very rich in epicatechin type B procyanidins, to have low galloylation %, and to have a high mean degree of polymerization of the proanthocyanidins with respect to the other analyzed wines.

H/D Exchange Processes in Flavonoids: Kinetics and Mechanistic Investigations.

Several classes of flavonoids, such as anthocyanins, flavonols, flavanols, and flavones, undergo a slow H/D exchange on aromatic ring A, leading to full deuteration at positions C(6) and C(8). Within the flavanol class, H-C(6) and H-C(8) of catechin and epicatechin are slowly exchanged in D2O to the corresponding deuterated analogues. Even quercetin, a relevant flavonol representative, shows the same behaviour in a D2O/DMSOd6 1:1 solution. Detailed kinetic measurements of these H/D exchange processes are here reported by exploiting the time-dependent changes of their peak areas in the 1H-NMR spectra taken at different temperatures. A unifying reaction mechanism is also proposed based on our detailed kinetic observations, even taking into account pH and solvent effects. Molecular modelling and QM calculations were also carried out to shed more light on several molecular details of the proposed mechanism.

Methyl Salicylate Glycosides in Some Italian Varietal Wines.

Glycosides are ubiquitous plant secondary metabolites consisting of a non-sugar component called an aglycone, attached to one or more sugars. One of the most interesting aglycones in grapes and wine is methyl salicylate (MeSA), an organic ester naturally produced by many plants, particularly wintergreens. To date, nine different MeSA glycosides from plants have been reported, mainly spread over the genera Gaultheria, Camellia, Polygala, Filipendula, and Passiflora. From a sensorial point of view, MeSA has a balsamic-sweet odor, known as Wintergreen. MeSA was found in Vitis riparia grapes, in Vitis vinifera sp. and in the Frontenac interspecific hybrid. We found that the MeSA glycosides content in Verdicchio wines and in some genetically related varieties (Trebbiano di Soave and Trebbiano di Lugana) was very high. In order to understand which glycosides were present in wine, the methanolic extract of Verdicchio wine was injected into a UPLC-Q-TOF-HDMS and compared to the extracts of different plants rich in such glycosides. Using pure standards, we confirmed the existence of two glycosides in wine: MeSA 2-O--d-glucoside and MeSA 2-O--d-xylopyranosyl (1-6) -d-glucopyranoside (gaultherin). For the first time, we also tentatively identified other diglycosides in wine: MeSA 2-O--l-arabinopyranosyl (1-6)--d-glucopyranoside (violutoside) and MeSA 2-O--d-apiofuranosyl (1-6)--d-glucopyranoside (canthoside A), MeSA 2-O--d-glucopyranosyl (1-6)-O--d-glucopyranoside (gentiobioside) and MeSA 2-O--l-rhamnopyranosyl (1-6)--d-glucopyranoside (rutinoside). Some of these glycosides have been isolated from Gaultheria procumbens leaves by preparative liquid chromatography and structurally annotated by 1H- and 13C-NMR analysis. Two of the peaks isolated from Gaultheria procumbens leaves, namely MeSA sambubioside and MeSA sophoroside, were herein observed for the first time. Six MeSA glycosides were quantified in 64 Italian white wines, highlighting the peculiar content and pattern in Verdicchio wines and related cultivars. The total concentration in bound and free MeSA in Verdicchio wines varied in the range of 456-9796 g/L and 5.5-143 g/L, respectively, while in the other wines the bound and free MeSA was below 363 g/L and 12 g/L, respectively. As this compound's olfactory threshold is between 50 and 100 g/L, our data support the hypothesis that methyl salicylate can contribute to the balsamic scent, especially in old Verdicchio wines.

Lipid Profiling and Stable Isotopic Data Analysis for Differentiation of Extra Virgin Olive Oils Based on Their Origin.

To differentiate extra virgin olive oils (EVOO) according to the origin of purchase, such as monocultivar Italian EVOO with protected denomination of origin (PDO) and commercially-blended EVOO purchased in supermarkets, a number of samples was subjected to the analysis of various lipid species by liquid chromatography/mass spectrometry (LC-ESI-MS/MS, LC-ESI-IT-MS) and proton nuclear magnetic resonance analysis (1H-NMR). Many putative chemical markers were extracted as differentiators by uni- and multivariate statistical analysis. Commercially-blended EVOO contained higher concentrations of the majority of minor lipids, including free fatty acids, their alkyl (methyl and ethyl) esters, monoglycerides, and diglycerides, which may be indicative of a higher degree of triglyceride lipolysis in these than in monocultivar PDO EVOO. Triterpenoids and particular TAG species were also found in higher proportions in the samples from the commercially-blended EVOO class, suggesting a possible influence of factors such as the cultivar and geographical origin. The largest differences between the classes were determined for the concentrations of uvaol and oleanolic acid. The results of the analysis by isotopic ratio mass spectrometry (IRMS) were reasonably consistent with the information about the geographical origin declared on the labels of the investigated EVOOs, showing considerable variability, which possibly also contributed to the differences in lipid composition observed between the two investigated classes of EVOO.

IsotopicLabelling: an R package for the analysis of MS isotopic patterns of labelled analytes.

MOTIVATION: Labelling experiments in biology usually make use of isotopically enriched substrates, with the two most commonly employed isotopes for metabolism being 2H and 13C. At the end of the experiment some metabolites will have incorporated the labelling isotope, to a degree that depends on the metabolic turnover. In order to propose a meaningful biological interpretation, it is necessary to estimate the amount of labelling, and one possible route is to exploit the fact that MS isotopic patterns reflect the isotopic distributions. RESULTS: We developed the IsotopicLabelling R package, a tool able to extract and analyze isotopic patterns from liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-MS (GC-MS) data relative to labelling experiments. This package estimates the isotopic abundance of the employed stable isotope (either 2H or 13C) within a specified list of analytes. AVAILABILITY AND IMPLEMENTATION: The IsotopicLabelling R package is freely available at https://github.com/RuggeroFerrazza/IsotopicLabelling CONTACTS: r.ferrazza@unitn.itSupplementary information: Supplementary data are available at Bioinformatics online.

Novel green algal isolates from the Egyptian hyper-arid desert oases: a polyphasic approach with a description of Pharao desertorum gen. et sp. nov. (Chlorophyceae, Chlorophyta).

The biodiversity of terrestrial algae is still grossly understudied, and African deserts in particular are barely touched in this respect. Here, four coccoid green algae from oases in the Western Desert of Egypt were characterized using a combination of morphotaxonomic, ecological and 18S rDNA data, with additional carotenoid and lipid analyses for two of the strains. Three strains were identified as affiliated with known taxa: Mychonastes sp., Asterarcys sp. (first report of this genus from a desert soil), and Stichococcus cf. deasonii. The fourth strain is proposed to represent a new cryptic genus Pharao gen. nov., with the type species P. desertorum sp. nov. The new taxon is sister to the clade of uncharacterized North American desert strains of Radiococcaceae (Chlorophyceae, Chlorophyta). The pigment profile of P. desertorum gen. et sp. nov. revealed carotenoids and chlorophylls typical of green algae. Bioorganic analysis showed a complex lipidome based on phospho- (PC), galacto- (MGDG and DGDG), betaine- (DGTS), and sulfoquinovosyl- (SQDG) membrane lipids, besides significant amounts of storage neutral lipids such as diacyl- (DAG) and triacylglycerols (TAG). The presence of saturated alkyl chains within all the membrane lipid classes in P. desertorum and Asterarcys sp. appears to reflect the need to maintain membrane fluidity and viscosity. In summary, African deserts likely still harbor new taxa to be described, and lipidomic analyses of such taxa may provide clues about their ability to survive in the extremely harsh desert habitats.

New Sulfur-Containing Polyarsenicals from the New Caledonian Sponge Echinochalina bargibanti.

Arsenicin A (C3H6As4O3) was isolated from the New Caledonian poecilosclerid sponge Echinochalina bargibanti, and described as the first natural organic polyarsenic compound. Further bioguided fractionation of the extracts of this sponge led us to isolate the first sulfur-containing organic polyarsenicals ever found in Nature. These metabolites, called arsenicin B and arsenicin C, are built on a noradamantane-type framework that is characterized by an unusual As⁻As bonding. Extensive NMR measurements, in combination with mass spectra, enabled the assignment of the structure for arsenicin B (C3H6As4S2) as 2. The scarcity of arsenicin C and its intrinsic chemical instability only allowed the collection of partial spectral data, which prevented the full structural definition. After the extensive computational testing of several putative structures, structure 3 was inferred for arsenicin C (C3H6As4OS) by comparing the experimental and density functional theory (DFT)-calculated ¹H and 13C NMR spectra. Finally, the absolute configurations of 2 and 3 were determined with a combined use of experimental and time-dependent (TD)-DFT calculated electronic circular dichroism (ECD) spectra and observed specific rotations. These findings pose great challenges for the investigation of the biosynthesis of these metabolites and the cycle of arsenic in Nature. Arsenicins B and C showed strong antimicrobial activities, especially against S. aureus, which is comparable to the reference compound gentamycin.

Anticancer Activity of Euplotin C, Isolated from the Marine Ciliate Euplotes crassus, Against Human Melanoma Cells.

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

TLC surface integrity affects the detection of alkali adduct ions in TLC-MALDI analysis.

Direct coupling of thin-layer chromatography (TLC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry allows fast and detailed characterization of a large variety of analytes. The use of this technique, however, presents great challenges in semiquantitative applications because of the complex phenomena occurring at the TLC surface. In our laboratory, we recently observed that the ion intensities of several alkali adduct ions were significantly different between the top and interior layer of the TLC plate. This indicates that the integrity of the TLC surface can have an important effect on the reproducibility of TLC- MALDI analyses. Graphical Abstract MALDI imaging reveals that surface integrity affects the detection of alkali adductions in TLC-MALDI.

Micro- versus nano-sized molecularly imprinted polymers in MALDI-TOF mass spectrometry analysis of peptides.

The integration of molecularly imprinted polymers (MIPs) with MALDI-TOF mass spectrometry (MS) combines MIP selectivity with MS sensitivity. Whether the size of the MIP material-micro versus nano-has an effect on the MS analysis was the object of the study. MIPs, targeting respectively the epitope peptide NR11 of cardiac troponin I and the peptide CK13 of human serum transferrin, were synthesized and characterized. The size-related performance of the MIP materials hyphenated with MALDI-TOF-MS analysis was studied by the incubation of the target peptide with the respective micro- or nano-MIP, followed by rinsing to remove non-specific deposition of the MIP to the MALDI target plate, co-crystallization with the organic matrix, and mass analysis. The quality of the MS analysis was assessed comparing the S/N of the mass peaks of the MIP-bound peptide to that of the same quantity of free peptide. Sweet spots and lower S/N (~ 1 order of magnitude) were observed for micro-MIP materials, while in the case of nano-MIP-bound peptide, the S/N was comparable to that of the free peptide, indicating higher compatibility of the nano-MIPs to MALDI-TOF-MS. The nano-MIP/MALDI-TOF-MS permitted the selective determination of the target peptide in real serum samples. Graphical abstract ᅟ.

Chemical Defense by Erythrolactones in the Euryhaline Ciliated Protist, Pseudokeronopsis erythrina.

Pseudokeronopsis erythrina produces three new secondary metabolites, erythrolactones A2, B2 and C2, and their respective sulfate esters (A1, B1, C1), the structures of which have been recently elucidated on the basis of NMR spectroscopic data coupled to high resolution mass measurements (HR-MALDI-TOF). An analysis of the discharge of the protozoan pigment granules revealed that the non-sulfonated erythrolactones are exclusively stored in these cortical organelles, which are commonly used by a number of ciliates as chemical weapons in offense/defense interactions with prey and predators. We evaluated the toxic activity of pigment granule discharge on a panel of free-living ciliates and micro-invertebrates, and the activity of each single purified erythrolactone on three ciliate species. We also observed predator-prey interactions of P. erythrina with unicellular and multicellular predators. Experimental results confirm that only P. erythrina cells with discharged pigment granules were preferentially or exclusively hunted and eaten by at least some of its predators, whereas almost all intact (fully pigmented) cells remained alive. Our results indicate that erythrolactones are very effective as a chemical defense in P. erythrina.

Natural Products among Brown Algae: The Case of Cystoseira schiffneri Hamel (Sargassaceae, Phaeophyceae).

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone (1) and a new isololiolide derivative named schiffnerilolide (2). The structure elucidation and the assignment of relative configurations of the isolated natural products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae belonging to genus Sargassum C. Agardh. Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigment analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes.