Centric Diatom Cyclotella Cryptica Research Articles

Fucoxanthin-Chlorophyll Protein Complexes of the Centric Diatom Cyclotella Meneghiniana Differ in Lhcx1 and Lhcx6_1 Content.

The ecological success of diatoms, key contributors to photosynthesis, is partly based on their ability to perfectly balance efficient light harvesting and photoprotection. Diatoms contain higher numbers of antenna proteins than vascular plants for light harvesting and for photoprotection. These proteins are arranged in fucoxanthin-chlorophyll protein (FCP) complexes. The number of FCP complexes, their subunit composition, and their interactions in the thylakoid membranes remain elusive in different diatoms. We used the recently available genome sequence of the centric diatom Cyclotella cryptica to analyze gene sequences for putative light-harvesting proteins in C. meneghiniana, and to elucidate the FCP complex composition. We analyzed two pools of FCP complexes that were trimeric (FCPa) and nonameric (FCPb). FCPa was composed of four different trimeric subtypes. Two different nonameric FCPb complexes were present. All were distinguished by their polypeptide composition and partly by pigmentation. With use of a milder purification method, two fractions composed of different FCP complexes were isolated. One was enriched in FCPs incorporating the photoprotective subunit Lhcx1, such as the newly identified nonameric FCPb2 and the major trimeric FCPa4 complex, which are predetermined to be involved in energy-dependent nonphotochemical quenching. The other fraction contained mainly FCPs that were devoid of Lhcx1, FCPa3, and FCPb1. Both fractions also included small amounts of trimeric FCPa complexes with the centric diatom-specific Lhcx protein, Lhcx6_1, as subunit. Thus, the antenna organization of centric diatoms, as well as the distribution of different photoprotective Lhcx proteins, differs from that of other diatoms, as well as from plants.

Association of Fucoxanthin Chlorophyll a/c-binding Polypeptides with Photosystems and Phosphorylation in the Centric Diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.

Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy

Antisera were raised against the C termini of three fucoxanthin chlorophyll a/c-binding polypeptides, Fcp2, Fcp4, and Fcp6, of the centric diatom Cyclotella cryptica. Immunogold electron microscopy of ultrathin-sectioned cells indicated that Fcp2 and Fcp4 are present in almost the same amounts, whereas approximately 8- to 10-fold less gold label was registered for Fcp6. Immunogold electron microscopy of freeze-fracture replicas of thylakoid membranes showed that the C termini of at least Fcp2 and Fcp4 were located in the thylakoid lumen, thus demonstrating a 3-dimensional structure similar to that already described for the chlorophyll a/b-binding light-harvesting polypeptides of higher plants.

Investigations on Transcript Sizes, Steady State mRNA Concentrations and Diurnal Expression of Genes Encoding Fucoxanthin Chlorophyll a/c Light Harvesting Polypeptides in the Centric Diatom Cyclotella cryptica

Abstract: The transcript sizes of fcp genes of Cyclotella cryptica, belonging to different fcp gene clusters and encoding different fucoxanthin chlorophyll a/c light harvesting polypeptides (Fcps), were determined. Furthermore, the diurnal expression of the fcp1/fcp2/fcp3/fcp5 gene cluster and the steady state mRNA concentrations of all gene clusters were investigated in response to light quality and quantity. The mRNAs of the gene cluster consisting of fcp1/fcp2/fcp3/fcp5 are approx. 950 bases in length, whereas those of the fcp4 and the fcp6/fcp7/fcp12 clusters are approx. 1050 (fcp4), 880 (fcp6/fcp7) and 1150 (fcp12) bases in lengths. The transcript levels of the fcp1/fcp2/fcp3/fcp5 gene cluster increased with the onset of light, reached a maximum around noon and dropped in the afternoon. The steady state mRNA concentrations of the fcp1/fcp2/fcp3/fcp5 gene cluster and of the fcp4 gene cluster were higher, when Cyclotella cryptica was grown in low light, while the steady state mRNA concentration of the fcp6/fcp7/fcp12 gene cluster increased under high light growth conditions. The steady state mRNA concentrations of all gene clusters were highest when Cyclotella cryptica was grown in red light, intermediate in green light, and lowest in blue light.

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.

Characterization of fcp4 and fcp12, Two Additional Genes Encoding Light Harvesting Proteins of Cyclotella cryptica (Bacillariophyceae) and Phylogenetic Analysis of this Complex Gene Family

Abstract: Two additional cDNA clones containing genes which encode fucoxanthin chlorophyll a/c binding proteins (Fcps) of the centric diatom Cyclotella cryptica have been sequenced. The first cDNA clone containing fcp12 had an insert size of 871 base pairs (bp). The open reading frame (ORF) of 693 bp corresponds to a precursor protein of 231 amino acids with a molecular weight (Mr) of 25 200. For the mature Fcp12, a protein of 196 amino acids with a Mr of 21 700 is proposed. The second cDNA clone contained the fragmentary fcp4 with an insert of 805 bp. The ORF of 492 bp corresponds to a polypeptide of 164 amino acids with a Mr of 18 050. Phylogenetic analyses revealed that the proteins Fcp1, Fcp2, Fcp3 and Fcp5 are closely related to the Fcps of other diatoms, whereas Fcp6, Fcp7 and Fcp12 share the highest homology to the Fcp of the haptophyte Isochrysis galbana and to the light inducible proteins LI818r3 and LI818 of Chlamydomonas reinhardtii and Chlamydomonas eugametos. The subunit Fcp4 revealed some homology with the red algal LH subunits LhcaR1 and LhcaR2 of Porphyridium cruentum.

Investigations on Gene Copy Number, Introns and Chromosomal Arrangement of Genes Encoding the Fucoxanthin Chlorophyll a/c-Binding Proteins of the Centric Diatom Cyclotella cryptica

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.

GENETIC TRANSFORMATION OF THE DIATOMS CYCLOTELLA CRYPTICA AND NAVICULA SAPROPHILA

ABSTRACT Two species of diatoms were genetically transformed by introducing plasmid vectors containing the Escherichia coli neomycin phosphotransferase II (nptII)gene. Expression of the bacterial nptII gene in the diatoms was achieved using the putative promoter and terminator sequences from the acetyl‐CoA carboxylase gene from the centric diatom Cyclotella cryptica T13L Reimann, Lewin, and Guillard. The vectors were introduced into C. cryptica and the pennate diatom Navicula saprophila NAVIC1 Lange‐Bertalot and Bonik by microprojectile bombardment. Putative transformants were selected based on their ability to grow in the presence of the antibiotic G418, and production of the neomycin phosphotransferase protein by the transformed cells was confirmed by western blotting. The foreign DNA integrated into one or more random sites within the genome of the transformed algal cells, often in the form of tandem repeats. This is the first report of reproducible, stable genetic transformation of a chlorophyll c‐containing alga.

Wirkung von Silatranen auf Wachstum, Sauerstoffentwicklung und Dunkelatmung von Cyclotella cryptica (Diatomeae) / The Effect of Silatranes on Growth, Photosynthetic Oxygen Evolution and Dark Respiration of Cyclotella cryptica (Diatomeae)

The effect of 5 silatranes RSi(OCH2CH2)3N, where R = C2HsO; C6H5; ClCH2 and RSi[OCH(CH3)CH2]3N, where R = C6H5, ClCH2 and tetraethoxysilane were studied on the growth, photosynthetic oxygen evolution and dark respiration of the marine centric diatom Cyclotella cryptica. Both silica-free and Si(OH)4 supplemented media were used. Only 1-ethoxy- silatrane was hydrolyzed by the diatom, giving sufficient quantities of Si(OH)4 to maintain growth without exogenous Si(OH)4. In this case the diatoms multiplied by a factor of 18 within 48 h. With silica in the medium, 1-phenylsilatrane, 1-phenyl-3,7,10-trimethylsilatrane, 1-chlormethylsilatrane and 1-chlorm ethyl-3,7,10-trimethylsilatrane exhibited only slightly inhibitory activity. In contrast, however, tetraethoxysilane (5 · 10-4 mol/1) inhibited growth almost completely. Respiration was unaffected by all the silatranes tested, but net photosynthetic oxygen evolution was reduced by approx. 50% by 1-chlormethylsilatrane and 1-chlormethyl- 3,7,10-trimethylsilatrane and almost completely by 1-phenylsilatrane and 1-phenyl-3,7,10-tri- methylsilatrane. In the absence of silica as nutrient in the medium, the silatranes hardly affected dark respiration within 24 h. Conversely, the same samples showed no photosynthetic oxygen evolution after 24 h, and 24 h later again a negative O2 balance was produced in the light. Different silatranes produced significant differences in the final oxygen balance.

Kalium-abhängige kieselsäureaufnahme im dunkeln bei Cyclotella cryptica

The changes in biochemical composition during a 9 h dark period of the marine centric diatom Cyclotella cryptica in a pure mineral medium are characterized by a decrease in the C content per dry weight by 4%, a decrease of H by 0.4%, an increase of Si by 5% and an increase of N by 0.4%. The dry weight of the cells per ml suspension remains constant during this period or increases even slightly. The increase in the silicate content of the cells by almost 50 % (from 23 to 34% per dry weight) is accompanied by an increase of the potassium content during the dark period by 100% (from 0.75 to 1.5% per dry weight). In a nitrogen deficient medium the same effects, slightly reduced, were observed. However, the N content of the cells did not remain constant, as expected, but decreased significantly between 3–6 h and increased again after a 9 h dark period. In a potassium free medium the silicic acid uptake in the dark is almost completely inhibited, but normal in a magnesium deficient medium. In a following light period, a reduced but significant silicic acid uptake is observed in the potassium-free medium. Decomposition of hexoses but not respiration is reduced in potassium-free medium. The data are discussed in relation to physiological cell stages which can be used to study the energetics of silicic acid uptake and polycondensation. The analyses support the previously described idea, that this diatom can assimilate a much higher quantity of silicate by use of a certain amount of stored carbon compounds.

Radiodünnschichtchromatographische untersuchungen mit 68Ge und 71Ge an extrakten aus Cyclotella cryptica

Summary Using 68 Ge and 71 Ge as tracers in silicate metabolism of the marine centric diatom Cyclotella cryptica , cell free extracts of labelled cells (supernatant 38,000 Xg) were analysed by thin layer chromatography. Only after hydrolysis at 100°C, a soluble fraction was found. In in vitro studies, a small fraction of the label could be bound to components of the cell free extract. Instead to components of cell free extracts, binding of 68 Ge(OH) 4 was possible also to the polyamino acid polyserine, but not to polymethionine. Several other low molecular weight substances with hydroxyl groups, such as mannitol, ascorbic acid, boric acid and isocitric acid were also used for binding studies without significant effects. The results are discussed in relation to the finding of a high serine and threonine content in the silicalemma of diatoms by other authors.

Stoffwechselregulation durch den Zellwandbaustein Kieselsäure: Polgrößenänderungen von a-Ketoglutarsäure, Aminosäuren und Nucleosidphosphaten

When the centric diatom Cyclotella cryptica is grown in a Si (OH) 4-free medium, the glutamic acid pool decreases within 3 hours to a third of the original value, whereas the aspartic acid pool is reduced by only about 20 per cent. The pools of nucleosid-triphosphates and of glycerol-1-phosphate remain unaffected during this time. The nucleosid-diphosphates pool decreases in the same way as that of aspartic acid. The decrease in the glutamic acid pool precedes the inhibition of total protein synthesis in Si (OH) 4-deficient cells, and a significant decrease in the a-ketoglutarate pool precedes the decrease of the glutamic acid content. Already within 60 minutes ofter incubation in a Si (OH) 4-free medium, the content of a-ketoglutarate is decreased to one third of the normal value. On the other hand, the acetyl CoA pathway (enhanced fatty acid synthesis) is not inhibited. The results suggest, that the Si (OH) 4-metabolism interferes with reactions between the condensing enzyme (acetyl-CoA and oxalacetate) and a-ketoglutarate. The delay between inhibition of protein- and RNA-synthesis and the different changes in the pools of amino acids and nucleosid-triphosphates resemble the regulation of the nucleosid-triphosphate pool and RNA-synthesis in amino acid starved strains of E. coli (EDLIN and NEUHARD) 1, though the primary causes are quite different.