Immuno-electron microscopic quantification of the fucoxanthin chlorophyll a/c binding polypeptides Fcp2, Fcp4, and Fcp6 of Cyclotella cryptica grown under low- and high-light intensities

The diatom Cyclotella cryptica was grown under low- and highintensity white light of 50 and 500 μmol photons m–2 s–1, respectively. Western immunoblotting showed that the diatom adapted its light-harvesting apparatus, giving rise to different amounts of distinct fucoxanthin chlorophyll a/c binding polypeptides (Fcp). The amount of Fcp2 was approximately two-fold higher under low-light than under high-light conditions, whereas the amount of Fcp6 increased four- to five-fold under high-light conditions. For Fcp4, no significant differences were detected in response to either light regime. Cells of Cyclotella grown under high- and low-light intensity were subjected to immunoelectron microscopy. Quantification of the gold label, expressed as gold particles per μm2, confirmed the results obtained by Western immunoblotting. Exposure to low light resulted in the detection of approximately six times more Fcp2-bound gold particles per μm2 in thylakoid membranes, whereas in cells grown under high light the number of Fcp6- bound gold particles increased ten-fold. For Fcp4, similar amounts of gold particles per μm2 were counted under the two light regimes. These immunocytochemical results confirmed molecular data derived from phylogenetic analyses of the sequences of genes encoding fucoxanthin chlorophyll a/c binding polypeptides (fcp genes) and from measurements of steady-state fcp mRNA concentrations. The results show that Fcp2 and Fcp6 accumulate under low- and high-light intensity, respectively, whereas Fcp4 seems to be constitutively synthesized. [Int Microbiol 2006; 9(1):29-36

Evidence of monomeric photosystem I complexes and phosphorylation of chlorophyll a/c-binding polypeptides in Chroomonas sp. strain LT (Cryptophyceae)

Thylakoid membranes of the cryptophyte Chroomonas sp. strain LT were solubilized with dodecyl-beta-maltoside and subjected to sucrose density gradient centrifugation. The four pigment protein complexes obtained were subsequently characterized by absorption and fluorescence spectroscopy, SDS-PAGE, and Western immunoblotting using antisera against the chlorophyll a/c-binding proteins of the marine cryptophyte Cryptomonas maculata and the reaction-center protein D2 of photosystem II of maize. Band 1 consisted mainly of free pigments, phycobiliproteins, and chlorophyll-a/c-binding proteins. Band 2 represented a major chlorophyll a/c-binding protein fraction. A mixture of photosystem II and photosystem I proteins comprised band 3, whereas band 4 was enriched in proteins of photosystem I. Western immunoblotting demonstrated the presence of chlorophyll a/c-binding proteins and their association with photosystem I in band 4. Phosphorylation experiments showed that chlorophyll a/c-binding proteins became phosphorylated. Negative staining electron microscopy of band B4 revealed photosystem I particles with dimensions of 22 nm. Our work showed that PSI-LHCI complexes of cryptophytes are similar to those of Chlamydomonas rheinhardtii, the diatom Phaeodactylum tricornutum, and higher plants

The membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria (formerly Cyanidium caldarium): biochemical and immunochemical characterization1Dedicated to Professor W.E. Krumbein on the occasion of his 60th birthday.1

AbstractThe membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria (formerly Cyanidium caldarium) could be isolated by gel-electrophoresis as a green band with an apparent molecular mass of about 20 kDa. The band had a long-wavelength absorption maximum at 672 nm and a fluorescence maximum (77 K) at 680 nm and reacted with an antibody against light-harvesting proteins of higher plant Photosystem I. Screening of thylakoid membranes with antisera directed against various chlorophyll a/b and chlorophyll a/c light-harvesting proteins indicated the existence of at least 4 distinct light-harvesting polypeptides with apparent molecular masses between 17 and 20 kDa. Isolation of Photosystem I and of a fraction enriched in Photosystem II showed that these polypeptides are exclusively bound to Photosystem I, thus forming a holocomplex which binds at least 205 molecules of chlorophyll a, and 33 and 37 molecules of zeaxanthin and β-carotene, respectively. Additionally, there is some evidence for the existence of a second Photosystem I pool without light-harvesting complexes. In-vitro translation experiments showed that at least two of the five polypeptides which constitute the membrane-intrinsic light-harvesting complex of Galdieria sulphuraria are translated from the poly(A)-enriched RNA fraction. They could be immunoprecipitated as preproteins being 3 to 4 kDa larger in size than the mature polypeptides

Differential circadian expression of genes fcp2 and fcp6 in Cyclotella cryptica

The steady-state mRNA concentrations of two fcp genes encoding fucoxanthin chlorophyll a/c light-harvesting polypeptides of the centric diatom Cyclotella cryptica were investigated over a 4-day period by RNA dot-blotting experiments. Before and during the first day of the experiment, the cultures were grown under a 12-h light/12-h dark regime. On the following 3 days, the algae were kept in darkness. On the first day, the steady-state mRNA concentration of fcp2 followed a diurnal pattern, with a maximum occurring around noon, approximately 6 h after the onset of light. The gene fcp6 also had a diurnal pattern on the first day. Its maximum, however, occurred immediately after the onset of light. During the subsequent incubation period in darkness, the diurnal pattern of expression of both fcp genes continued, thus demonstrating that their steady-state mRNA concentrations oscillated in a circadian manner. [Int Microbiol 2004; 7(2):127–131

Vertical migration behaviour of diatom assemblages of Wadden Sea sediments (Dangast, Germany): a study using cryo-scanning electron microscopy

The vertical migration behaviour of diatom assemblages inhabiting Wadden Sea sediments near Dangast (Germany) was investigated using cryoscanning electron microscopy. The diatom assemblages were dominated by small Navicula species. Intertidal sediments which were located at different distances from the high tide level or stayed submerged even throughout low tides were chosen. Samples were prepared and cryofixed in the field. Sampling was restricted to three sets: (i) before the onset of vertical migration, (ii) 3 to 5 h after the onset of vertical migration, and (iii) before the area became flooded again or just prior to dusk. The diatom assemblages inhabiting the different types of sediments did not always show the same response. When the tidal cycle exposed the sediment surfaces during the night cell densities increased in the early morning hours with the onset of light. Later on, although the photon flux density was still increasing, cell densities stayed constant or decreased before the water flooded the areas around noon. In experiments in which the water drained off around noon and the areas became exposed throughout the entire afternoon, cell densities increased even up to dusk when the photon flux density had dropped to values below 20 μM photons m-2s-1. In an experiment in which the last sampling occured at 10.15 pm, when the photon flux density had already declined below 10 μM photons m-2s-1, cell densities had decreased to lower values. This was ca. 1 h before the area was flooded again. Finally, cryo-scanning electron microscopy revealed frequently occuring micropatches of diatom assemblages which could be differentiated into typical areas of lower and higher cell densities further complicating the pattern of light or water cover induced movements

A methodological approach to investigate steady state fucoxanthin chlorophyll a/c binding protein mRNA levels in Wadden Sea sediments

A method was established to investigate the steady state levels of mRNAs from genes encoding fucoxanthin chlorophyll a/c binding proteins (Fcp) of diatoms in situ. During the study, which was performed withWadden Sea sediments from the German North Sea shore near Dangast, oxygenic photosynthesis was carried out mainly by pennate diatoms. Field samples were taken after tidal exposure from dawn up to late afternoon at 2-hourly intervals, and frozen in liquid nitrogen. In the laboratory, total RNA was isolated by isopycnic ultracentrifugation in caesium chloride gradients. Yields of approximately 10–300 μg RNA per gram wet sediment were obtained. Defined amounts of total RNA were blotted onto nylon membranes and hybridised with probes against the fcp2 and 18S rDNA genes of Cyclotella cryptica. To estimate the steady state amount of fcp mRNAs, fcp signal intensities were normalized to the signal intensities obtained from hybridisation to an 18S rDNA gene probe. In the two time-course studies performed to demonstrate the applicability of the method, the steady state levels of fcp mRNA increased up to 12-fold with the onset of light, reaching a maximum 6–8 h after sunrise before they decreased again. Possible reasons for this time-course are discussed

The enigmatic nucleus of the marine dinoflagellateProrocentrum cordatum

The marine, bloom-forming dinoflagellateProrocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ∼4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specificnucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattenednucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (∼0.4-6.7 μm3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specificprocedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight)mass spectrometers, respectively. This allowed identificationof 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specificnuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differingfrom those of typical eukaryotic cells

Changes in the photosynthetic apparatus of diatoms in response to low and high light intensities

The centric diatom Cyclotella cryptica and two strains of the pennate diatom Phaeodactylum tricornutum were grown under low and high light intensities (300 lux and 3,000 lux) over 4—6 weeks. Growth was monitored by repetitive cell count. The culture media were replaced weekly to avoid morphological and biochemical alterations caused by nutrient depletion. The ultrastructure of the cells was examined by transmission electron microscopy. Alterations in the light-harvesting antenna systems were investigated by Western immunoblotting. Both diatoms reduced the plastid area, i.e. decreased the amount of thylakoid lamellae, under high light intensity. The thylakoids still ran in groups of three with parallel orientation within the chloroplasts. The girdle band lamellae were not affected. The amounts of storage compounds and vacuoles increased. SDS-PAGE of total cell protein followed by Western immunoblotting with antisera directed against subunits of the light-harvesting antenna systems of C. cryptica (cc-antiserum) and the cryptophyte Cryptomonas maculata (cmac-antiserum) revealed that both diatoms reduced the amount of antenna polypeptides under increased light intensity. The cc-antiserum immunodecorated two bands with relative molecular masses (Mr) of 18,000 and 22,000 in C. cryptica. Both decreased under high light conditions to 67.2 ± 6.1%. Five to seven bands in the Mr range of 14,000—27,000 were recognized in P. tricornutum. They decreased to 83 ± 5.3%. Furthermore, the immunolabeling pattern for both strains differed under the two light regimes. The cmac-antiserum immunodecorated two polypeptides with Mr of 24,000 and 23,000 in C. cryptica, while both strains of P. tricornutum had five polypeptides in the Mr range of 14,000—24,000 that showed some differences in staining intensities between the two strains and in response to the light intensity applied