THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

Isolation of humic acid from the brown algaPilayella littoralis

A standard humic acid extraction procedure has been used to isolate dark brown organic residues from samples of the macroscopic marine brown algaPilayella littoralis. The residues are insoluble in water, but soluble at high pH, and are similar in elemental composition, ash content, UV-visible, IR, PMR and X-Ray fluorescence spectra, X-Ray diffractograms and scanning electron micrographs to residues of a humic acid isolated from municipal compost. These results indicate thatPilayella produces humic acids.Author for correspondence     

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.     

Effect of exogenous gibberellin A4/7 on tracheid production, longitudinal growth and the levels of indole‐3‐acetic acid and gibberellins A4, A7 and A9 in the terminal shoot of Pinus sylvestris seedlings

Synchronously dividing cultures of the unicellular green alga Scenedesmus obtusiusculus were cultivated for 24 or 70 h in medium high (1000 μM) or low (60 μM) in phosphorus. Aliquots of AlCl₃ (0, 37, 74, 111, 148, 185, or 222 μmol) were added daily to 1 l cell suspension at the end of the cell division phase. Algae were also grown in media with different pH, adjusted with HCl, in the absence of AlCl₃.
Effects of Al on cell metabolism vary with the intracellular Al concentration and with the concentration of Al available per cell. When the concentration of phosphorus is low, internal concentrations of Al are high and the chlorophyll content and the net dry matter production per cell increase, whereas the photosynthesis and the cell division are increased. Presence of Al in a low P medium decreases the pH of the medium down to 4.5. There are only small effects of Al in the presence of P, due to precipitation of most of the Al with P in the medium.
Despite the Al-induced decrease of the pH of the culture medium, effects caused by Al cannot be explained as a pH effect. Instead, the Al effect may, at least to some extent, be related to a decrease in availability of P in the metabolism, due to formation of aluminium phosphate inside the cell.     

Actin and spectrin-like (Mr= 260–240 000) proteins in gregarines

In Gregarina blaberae a M_r = 47 000 and a M_r = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti-actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β-spectrin, respectively. The M_r = 47 000 actin-like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M_r = 260–240 000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin-like proteins is stage-dependent. Visualization of the M_r = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell-shape and the cell motility systems in gregarines. The presence of spectrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the M_r = 260–240 000 form.     

Structure and assembly of the cortical microtubule cytoskeleton in the green alga,Boodlea coacta

Summary Examination was made of the structure and assembly of the cortical microtubule (MT) cytoskeleton in the coenocytic green algaBoodlea coacta (Dickie) Murray et De Toni by immunofluorescence microscopy. Cortical MTs inBoodlea protoplasts are arranged randomly but some show a meridional arrangement within 6 h after protoplast formation. At 6–9 h such MTs become highly concentrated and parallel to each other in certain areas. At 12 h the concentration is uniformly high throughout the cell, indicating the completion of high density meridional arrangement of cortical MTs. Cortical MTs exhibiting a high density, meridional arrangement show characteristic disassembly by treatment with 10 M amiprophos-methyl (APM) or cold treatment (0 °C). Disassembly occurs by each MT unit at positions skipping 30–40 m in the transverse direction, and neighboring MTs subsequently disassemble to form MT groups. Each group becomes slender and then disappears completely within the following 24 h. The meridional arrangement of cortical MTs is disrupted by N-ethylmaleimide (NEM) accompanied by a remarkable reduction in density. The remaining MTs form groups at 30–40 m intervals from each other, as also occurs with drug or cold treatment, but disruption and density return to normal levels following removal of NEM. It appears that there are meridionally oriented channels, anchor-rich and anchor-poor, in the plasma membrane. The channels could be distributed alternately and anchors could be deposited in a cross-linking manner with cortical MTs to form a stable cortical MT-cytoskeleton. MTs comprising the cortical MT cytoskeleton could be oriented by meridionally oriented channels of anchors which are distributed following establishment of cell polarity.Abbreviations APM amiprophos-methyl - MT microtubule - MTOC microtubule organizing center - NEM N-ethylrnaleimide     

Effects of some factors on protoplast formation of a microalga,Prototheca zopfii

The effects of experimental factors on protoplast formation of Prototheca zopfii Kru¨ger in 0.85 m NaCl using Macerozyme R-200 were studied based on a fractional factorial experimental design. The rate of protoplast formation was mainly affected by the incubation temperature and the age of algal cells. The optimal condition for the maximum protoplast yield was determined based on a response surface model. These were: mid-logarithmic phase cells and Macerozyme concentration of 4% at a temperature of 35°C.