Identification of promoter targets by Aureochrome 1a in the diatom Phaeodactylum tricornutum.

Aureochromes (AUREOs) are unique blue light receptors and transcription factors found only in stramenopile algae. While each of the four AUREOs identified in the diatom Phaeodactylum tricornutum may have a specific function, PtAUREO1a has been shown to have a strong impact on overall gene regulation, when light changes from red to blue light conditions. Despite its significance, the molecular mechanism of PtAUREO1a is largely unexplored. To comprehend the overall process of gene regulation by PtAUREO1a, we conducted a series of in vitro and in vivo experiments, including pull-down assays, yeast one-hybrid experiments, and phenotypical characterization using recombinant PtAUREOs and diatom mutant lines expressing a modified PtAureo1a gene. We describe the distinct light absorption properties of four PtAUREOs and the formation of all combinations of their potential dimers. We demonstrate the capability of PtAUREO1a and 1b to activate the genes, diatom-specific cyclin 2, PtAureo1a, and PtAureo1c under both light and dark conditions. Using mutant lines expressing a modified PtAUREO1a protein with a considerably reduced light absorption, we found novel evidence that PtAUREO1a regulates the expression of PtLHCF15, which is essential for red light acclimation. Based on current knowledge, we present a working model of PtAUREO1a gene regulation properties.

Isolation of fucoxanthin chlorophyll protein complexes of the centric diatom Thalassiosira pseudonana associated with the xanthophyll cycle enzyme diadinoxanthin de-epoxidase.

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

[COVID-19: Collateral damage in head and neck oncology and preventive measures for future pandemics]. / COVID-19: Kollateralschäden in der Kopf-Hals-Onkologie und Präventivmaßnahmen für künftige Pandemien.

The start of the COVID-19 pandemic led to enormous challenges for global healthcare, as capacities and resources had to be made available quickly for the treatment of COVID-19 patients. As a result, restrictions had to be accepted, especially in the care of oncological patients. The collateral damage of these limitations inevitably also affects patients with head and neck cancer. This review article summarizes the development of tumor incidences during the pandemic, internationally developed guidelines for the care of patients with head and neck cancer and studies on the delay in oncological therapies and mortality. In addition, the effects on the mental health of the patients, the psychosocial consequences and ethical issues are examined. In perspective, preventive measures for such negative collateral effects in future pandemics are discussed using the example of a concept for application software (app)-based digital care for patients with head and neck cancer.

Meilensteine in der Algenbiotechnologie.

Recent progress in algal biotechnology has identified new products based on their broad evolutionary origin. Novel metabolites were found for pharmacy, food industry, medicine e.g. tumor suppression and antibiotics. However, sustainable and economical algal production for crude oil replacement is limited by extremely low space time yields in photobioreactors. The consequences are a high energy burden for mass flow dependent processes and the need of space being in conflict with sustainable landscape management. New concepts using algae not as biomass producers but as living catalysts may open new options.

Heterologous Lactate Synthesis in Synechocystis sp. Strain PCC 6803 Causes a Growth Condition-Dependent Carbon Sink Effect.

Cyanobacteria are considered promising hosts for product synthesis directly from CO2 via photosynthetic carbon assimilation. The introduction of heterologous carbon sinks in terms of product synthesis has been reported to induce the so-called "carbon sink effect," described as the release of unused photosynthetic capacity by the introduction of additional carbon. This effect is thought to arise from a limitation of carbon metabolism that represents a bottleneck in carbon and electron flow, thus enforcing a downregulation of photosynthetic efficiency. It is not known so far how the cellular source/sink balance under different growth conditions influences the extent of the carbon sink effect and in turn product formation from CO2, constituting a heterologous carbon sink. We compared the Synechocystis sp. strain PCC 6803 wild type (WT) with an engineered lactate-producing strain (SAA023) in defined metabolic states. Unexpectedly, high-light conditions combined with carbon limitation enabled additional carbon assimilation for lactate production without affecting biomass formation. Thus, a strong carbon sink effect only was observed under carbon and thus sink limitation, but not under high-sink conditions. We show that the carbon sink effect was accompanied by an increased rate of alternative electron flow (AEF). Thus, AEF plays a crucial role in the equilibration of source/sink imbalances, presumably via ATP/NADPH balancing. This study emphasizes that the evaluation of the biotechnological potential of cyanobacteria profits from cultivation approaches enabling the establishment of defined metabolic states and respective quantitative analytics. Factors stimulating photosynthesis and carbon fixation are discussed. IMPORTANCE Previous studies reported various and differing effects of the heterologous production of carbon-based molecules on photosynthetic and growth efficiency of cyanobacteria. The typically applied cultivation in batch mode, with continuously changing growth conditions, however, precludes a clear differentiation between the impact of cultivation conditions on cell physiology and effects related to the specific nature of the product and its synthesis pathway. In this study, we employed a continuous cultivation system to maintain defined source/sink conditions and thus metabolic states. This allowed a systematic and quantitative analysis of the effect of NADPH-consuming lactate production on photosynthetic and growth efficiency. This approach enables a realistic evaluation of the biotechnological potential of engineered cyanobacterial strains. For example, the quantum requirement for carbon production was found to constitute an excellent indicator of the source/sink balance and thus a key parameter for photobioprocess optimization. Such knowledge is fundamental for rational and efficient strain and process development.

The potential of multispectral imaging flow cytometry for environmental monitoring.

Environmental monitoring involves the quantification of microscopic cells and particles such as algae, plant cells, pollen, or fungal spores. Traditional methods using conventional microscopy require expert knowledge, are time-intensive and not well-suited for automated high throughput. Multispectral imaging flow cytometry (MIFC) allows measurement of up to 5000 particles per second from a fluid suspension and can simultaneously capture up to 12 images of every single particle for brightfield and different spectral ranges, with up to 60x magnification. The high throughput of MIFC has high potential for increasing the amount and accuracy of environmental monitoring, such as for plant-pollinator interactions, fossil samples, air, water or food quality that currently rely on manual microscopic methods. Automated recognition of particles and cells is also possible, when MIFC is combined with deep-learning computational techniques. Furthermore, various fluorescence dyes can be used to stain specific parts of the cell to highlight physiological and chemical features including: vitality of pollen or algae, allergen content of individual pollen, surface chemical composition (carbohydrate coating) of cells, DNA- or enzyme-activity staining. Here, we outline the great potential for MIFC in environmental research for a variety of research fields and focal organisms. In addition, we provide best practice recommendations.

Crossing and selection of Chlamydomonas reinhardtii strains for biotechnological glycolate production.

As an alternative to chemical building blocks derived from algal biomass, the excretion of glycolate has been proposed. This process has been observed in green algae such as Chlamydomonas reinhardtii as a product of the photorespiratory pathway. Photorespiration generally occurs at low CO2 and high O2 concentrations, through the key enzyme RubisCO initiating the pathway via oxygenation of 1.5-ribulose-bisphosphate. In wild-type strains, photorespiration is usually suppressed in favour of carboxylation due to the cellular carbon concentrating mechanisms (CCMs) controlling the internal CO2 concentration. Additionally, newly produced glycolate is directly metabolized in the C2 cycle. Therefore, both the CCMs and the C2 cycle are the key elements which limit the glycolate production in wild-type cells. Using conventional crossing techniques, we have developed Chlamydomonas reinhardtii double mutants deficient in these two key pathways to direct carbon flux to glycolate excretion. Under aeration with ambient air, the double mutant D6 showed a significant and stable glycolate production when compared to the non-producing wild type. Interestingly, this mutant can act as a carbon sink by fixing atmospheric CO2 into glycolate without requiring any additional CO2 supply. Thus, the double-mutant strain D6 can be used as a photocatalyst to produce chemical building blocks and as a future platform for algal-based biotechnology. KEY POINTS: • Chlamydomonas reinhardtii cia5 gyd double mutants were developed by sexual crossing • The double mutation eliminates the need for an inhibitor in glycolate production • The strain D6 produces significant amounts of glycolate with ambient air only.

Influence of the compatible solute sucrose on thylakoid membrane organization and violaxanthin de-epoxidation.

MAIN CONCLUSION: The compatible solute sucrose reduces the efficiency of the enzymatic de-epoxidation of violaxanthin, probably by a direct effect on the protein parts of violaxanthin de-epoxidase which protrude from the lipid phase of the thylakoid membrane. The present study investigates the influence of the compatible solute sucrose on the violaxanthin cycle of higher plants in intact thylakoids and in in vitro enzyme assays with the isolated enzyme violaxanthin de-epoxidase at temperatures of 30 and 10 °C, respectively. In addition, the influence of sucrose on the lipid organization of thylakoid membranes and the MGDG phase in the in vitro assays is determined. The results show that sucrose leads to a pronounced inhibition of violaxanthin de-epoxidation both in intact thylakoid membranes and the enzyme assays. In general, the inhibition is similar at 30 and 10 °C. With respect to the lipid organization only minor changes can be seen in thylakoid membranes at 30 °C in the presence of sucrose. However, sucrose seems to stabilize the thylakoid membranes at lower temperatures and at 10 °C a comparable membrane organization to that at 30 °C can be observed, whereas control thylakoids show a significantly different membrane organization at the lower temperature. The MGDG phase in the in vitro assays is not substantially affected by the presence of sucrose or by changes of the temperature. We conclude that the presence of sucrose and the increased viscosity of the reaction buffers stabilize the protein part of the enzyme violaxanthin de-epoxidase, thereby decreasing the dynamic interactions between the catalytic site and the substrate violaxanthin. This indicates that sucrose interacts with those parts of the enzyme which are accessible at the membrane surface of the lipid phase of the thylakoid membrane or the MGDG phase of the in vitro enzyme assays.

Phosphine-Stabilized Germasilenylidene: Source for a Silicon-Atom Transfer.

A phosphine-stabilized germasilenylidene is synthesized following the pathway of SiCl4 oxidative addition at a germylene-phosphine Lewis pair. Low-temperature reduction using {(MesNacnac)Mg}2 resulted in a chlorosilylene intermediate and finally a molecule exhibiting a Ge═Si: motif. Inside the chelating phosphine-germylene, a low-valent silicon atom is stabilized and was transferred to diazabutadiene to give N-heterocyclic silylenes. Because of the high reactivity of the phosphine-stabilized germasilenylidene, a reaction of two Ge═Si: units was found to yield a Si2Ge2-ring molecule exhibiting a germasilene substituted with a silylene.

Tipifarnib in recurrent, metastatic HRAS-mutant salivary gland cancer.

BACKGROUND: To the authors' knowledge, there are no approved therapies for recurrent, metastatic (R/M) salivary gland carcinoma (SGC), but molecularly targeted therapies warrant ongoing investigation. In the current study, the authors have reported on the efficacy of tipifarnib in patients with aggressive HRAS-mutant, R/M SGC. METHODS: The current prospective, nonrandomized, multicenter, international cohort study involved 8 centers and was conducted from May 2015 to June 2019. The median follow-up was 22 months (range, 6-55 months). Subjects with HRAS-mutant R/M SGC (any histology) and disease progression within the last 6 months were enrolled. Tipifarnib was dosed orally twice daily. The authors determined the objective response rate using Response Evaluation Criteria in Solid Tumors (version 1.1), duration of response, and molecular predictors of response. RESULTS: A total of 13 patients with R/M SGC were enrolled; all had received prior systemic therapy (1-3 regimens). One objective response was observed; an additional 7 of 12 evaluable patients (58%) had stable disease as their best response with a median duration of 9 months (range, 3-14 months). Five of 7 patients had >10% tumor regression and 6 of 7 had stable disease lasting >6 months. Q61R was the most frequent activating HRAS mutation noted (7 of 13 patients; 54%), but gene variant and allele frequency did not correlate with outcomes. The median progression-free survival was 7 months (95% confidence interval, 5.9-10.1 months), and the median overall survival was 18 months (95% confidence interval, 9.6-22.4 months) with approximately 58.6% of patients alive at 1 year. Survival was similar regardless of HRAS mutant variant or co-occurring PIK3CA alterations. No participant discontinued treatment because of toxicity. CONCLUSIONS: Tipifarnib resulted in modest clinical activity with a promising disease control rate among patients with HRAS-mutant, R/M SGC who developed disease progression within the last 6 months.

Pre-purification of diatom pigment protein complexes provides insight into the heterogeneity of FCP complexes.

BACKGROUND: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. RESULTS: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. CONCLUSION: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

Quantitative macromolecular patterns in phytoplankton communities resolved at the taxonomical level by single-cell Synchrotron FTIR-spectroscopy.

BACKGROUND: Technical limitations regarding bulk analysis of phytoplankton biomass limit our comprehension of carbon fluxes in natural populations and, therefore, of carbon, nutrients and energy cycling in aquatic ecosystems. In this study, we took advantage of Synchrotron FTIR micro-spectroscopy and the partial least square regression (PLSr) algorithm to simultaneously quantify the protein, lipid and carbohydrate content at the single-cell level in a mock phytoplankton community (composed by a cyanobacterium, a green-alga and a diatom) grown at two temperatures (15 °C and 25 °C). RESULTS: The PLSr models generated to quantify cell macromolecules presented high quality fit (R2 ≥ 0.90) and low error of prediction (RMSEP 2-6% of dry weight). The regression coefficients revealed that the prediction of each macromolecule was not exclusively dependent on spectral features corresponding to that compound, but rather on all major macromolecular pools, reflecting adjustments in the overall cell carbon balance. The single-cell analysis, studied by means of Kernel density estimators, showed that the modes of density distribution of macromolecules were different at 15 °C and 25 °C. However, a substantial proportion of cells was biochemically identical at the two temperatures because of population heterogeneity. CONCLUSIONS: The spectroscopic approach presented in this study allows the quantification of macromolecules in single phytoplankton cells. This method showed that population heterogeneity most likely ensures a backup of non-acclimated cells that may rapidly exploit new favourable niches. This finding may have important consequences for the ecology of phytoplankton populations and shows that the "average cell" concept might substantially limit our comprehension of population dynamics and biogeochemical cycles in aquatic ecosystems.

Glycolate from microalgae: an efficient carbon source for biotechnological applications.

Glycolate is produced in autotrophic cells under high temperatures and Ci -limitation via oxygenation of ribulose-1,5-bisphosphate. In unicellular algae, glycolate is lost via excretion or metabolized via the C2 cycle by consuming reductants, ATP and CO2 emission (photorespiration). Therefore, photorespiration is an inhibitory process for biomass production. However, cells can be manipulated in a way that they become glycolate-producing 'cell factories', when the ratio carboxylation/oxygenation is 2. If under these conditions the C2 cycle is blocked, glycolate excretion becomes the only pathway of photosynthetic carbon flow. The study aims to proof the biotechnological applicability of algal-based glycolate excretion as a new biotechnological platform. It is shown that cells of Chlamydomonas can be cultivated under specific conditions to establish a constant and long-term stable glycolate excretion during the light phase. The cultures achieved a high efficiency of 82% of assimilated carbon transferred into glycolate biosynthesis without losses of function in cell vitality. Moreover, the glycolate accumulation in the medium is high enough to be directly used for microbial fermentation but does not show toxic effects to the glycolate-producing cells.

15N photo-CIDNP MAS NMR on both photosystems and magnetic field-dependent 13C photo-CIDNP MAS NMR in photosystem II of the diatom Phaeodactylum tricornutum.

Diatoms contribute about 20-25% to the global marine productivity and are successful autotrophic players in all aquatic ecosystems, which raises the question whether this performance is caused by differences in their photosynthetic apparatus. Photo-CIDNP MAS NMR presents a unique tool to obtain insights into the reaction centres of photosystems (PS), by selective enhancement of NMR signals from both, the electron donor and the primary electron acceptor molecules. Here, we present the first observation of the solid-state photo-CIDNP effect in the pennate diatoms. In comparison to plant PSs, similar spectral patterns have been observed for PS I at 9.4 T and PS II at 4.7 T in the PSs of Phaeodactylum tricornutum. Studies at different magnetic fields reveal a surprising sign change of the 13C photo-CIDNP MAS NMR signals indicating an alternative arrangement of cofactors which allows to quench the Chl a donor triplet state in contrast to the situation in plant PS II. This unusual quenching mechanism is related to a carotenoid molecule in close vicinity to the Chl a donor. In addition to the photo-CIDNP MAS NMR signals arising from the donor and the primary electron acceptor cofactors, a complete set of signals of the imidazole ring ligating to the magnesium of Chl a can be observed.

Monitoring cellular C:N ratio in phytoplankton by means of FTIR-spectroscopy.

Statistical growth rate modelling can be applied in a variety of ecological and biotechnological applications. Such models are frequently based on Monod or Droop equations and, especially for the latter, require reliable determination of model input parameters such as C:N quotas. Besides growth rate modelling, a C:N quota quantification can be useful for monitoring and interpretation of physiological acclimation to abiotic and biotic disturbances (e.g., nutrient limitations). However, as high throughput C:N quota determination is difficult to perform, alternatives need to be established. Fourier-transformed infrared (FTIR) spectroscopy is used to analyze a variety of biochemical, chemical, and physiological parameters in phytoplankton. Hence, a quantification of the C:N quota should also be feasible. Therefore, using FTIR spectroscopy, six phytoplankton species from among different phylogenetic groups have been analyzed to determine the effect of nutrient limitation on C:N quota patterns. The typical species-specific response to increasing nitrogen limitation was an increase in the C:N quota. Irrespective of this species specificity, we were able to develop a reliable multi-species C:N quota prediction model based on FTIR spectroscopy using the partial least square regression (PLSR) algorithm. Our data demonstrate that the PLSR approach is more robust in C:N quota quantification (R2  = 0.93) than linear correlation of C:N quota versus growth rate (R2 ranges from 0.74 to 0.86) or biochemical information based on FTIR spectra (R2 ranges from 0.82 to 0.89). This accurate prediction of C:N values may support high throughput measurements in a broad range of future approaches.

The artificial humic substance HS1500 does not inhibit photosynthesis of the green alga Desmodesmus armatus in vivo but interacts with the photosynthetic apparatus of isolated spinach thylakoids in vitro.

Humic substances (HSs) can influence the growth and composition of freshwater phytoplankton assemblage. Since HSs contain many phenolic and quinonic moieties and cause growth reductions in eco-physiological field experiments, HSs are considered photosystem II herbicides. To test this specific mode of action in vivo and in vitro, respectively, we used intact cells of the green alga Desmodesmus armatus, as well as thylakoids isolated from spinach (Spinacia oleracea) as a model system for the green algal chloroplast. Photosynthetic electron transport was measured as oxygen evolution and variable chlorophyll fluorescence. The in vivo effect of the artificial humic substance HS1500 on algae consisted of no impact on photosynthesis-irradiance curves of intact green algae compared to untreated controls. In contrast, addition of HS1500 to isolated thylakoids resulted in light-induced oxygen consumption (Mehler reaction) as an in vitro effect. Fluorescence induction kinetics of HS-treated thylakoids revealed a large static quenching effect of HS1500, but no inhibitory effect on electron transport. For the case of intact algal cells, we conclude that the highly hydrophilic and rather large molecules of HS1500 are not taken up in effective quantities and, therefore, cannot interfere with photosynthesis. The in vitro tests show that HS1500 has no inhibitory effect on photosystem II but operates as a weak, oxygen-consuming Hill acceptor at photosystem I. Hence, the results indicate that eco-physiological field experiments should focus more strongly on effects of HSs on extracellular features, such as reducing and red-shifting the underwater light field or influencing nutrient availability by cation exchange within the plankton network.

Contrast-Enhanced Ultrasound in Pulmonary Lymphoma: A Small Pilot Study.

Here, we describe the appearance and pattern of pulmonary lymphoma on B-mode imaging and with contrast-enhanced ultrasound (CEUS). From July 2009 to December 2015, 6 patients with histologically or cytologically confirmed lymphoma of the lung were examined by B-mode imaging, followed by CEUS. A retrospective analysis of the imaging data was performed with respect to the time to enhancement, pulmonary artery (PA) and bronchial artery, echogenicity (hypoechoic, isoechoic, or hyperechoic), and homogeneity (homogeneous or inhomogeneous) of the contrast enhancement. On B-mode imaging, all 6 pulmonary lymphoma lesions were hypoechoic. Five cases had PA enhancement, and 1 case had bronchial artery enhancement on CEUS imaging. Strikingly, all 6 patients had isoechoic arterial contrast enhancement. In the parenchymal phase, 3 of the lymphoma lesions showed hypoechoic contrast enhancement, and 3 showed isoechoic enhancement. Pulmonary lymphomas are hypoechoic on B-mode imaging. With CEUS, all patients had predominant PA contrast enhancement in the arterial phase with variable parenchymal contrast enhancement. Thus, definite differentiation from other malignant or benign pulmonary lesions cannot be achieved by CEUS.

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport.

BACKGROUND: The preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction. RESULTS: Spectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to diatoxanthin and diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants. CONCLUSIONS: The present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically.

Phytoplankton growth rate modelling: can spectroscopic cell chemotyping be superior to physiological predictors?

Climate change has a strong impact on phytoplankton communities and water quality. However, the development of robust techniques to assess phytoplankton growth is still in progress. In this study, the growth rate of phytoplankton cells grown at different temperatures was modelled based on conventional physiological traits (e.g. chlorophyll, carbon and photosynthetic parameters) using the partial least square regression (PLSR) algorithm and compared with a new approach combining Fourier transform infrared-spectroscopy and PLSR. In this second model, it is assumed that the macromolecular composition of phytoplankton cells represents an intracellular marker for growth. The models have comparable high predictive power (R2 > 0.8) and low error in predicting new observations. Interestingly, not all of the predictors present the same weight in the modelling of growth rate. A set of specific parameters, such as non-photochemical fluorescence quenching (NPQ) and the quantum yield of carbon production in the first model, and lipid, protein and carbohydrate contents for the second one, strongly covary with cell growth rate regardless of the taxonomic position of the phytoplankton species investigated. This reflects a set of specific physiological adjustments covarying with growth rate, conserved among taxonomically distant algal species that might be used as guidelines for the improvement of modern primary production models. The high predictive power of both sets of cellular traits for growth rate is of great importance for applied phycological studies. Our approach may find application as a quality control tool for the monitoring of phytoplankton populations in natural communities or in photobioreactors.

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

MAIN CONCLUSION: A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.