Glutamate agonists from marine algae

The occurrence of three glutamate agonists — glutamic acid, D-homocysteic acid and kainic acid — in a spontaneous mutant of Palmaria palmata is reported. Glutamic acid and D-homocysteic acid, but not kainic acid, were found in the wild-type plant. The closely related glutamate agonist, domoic acid, was found in the red alga Chondria baileyana and in the diatom Nitzschia pungens forma multiseries. In the diatom, domoic acid can build up to high levels in excess of 3% (dry wt.), making N. pungens a potential commercial source of this neuroactive amino acid. Information is also presented on the distribution, chemistry and biological activity of neuroactive amino acids from algae, and a possible biogenic relationship among kainoid metabolites is discussed. author for correspondence     

Feeding strategy of the sacoglossan opisthobranch <Emphasis Type="Italic">Oxynoe olivacea</Emphasis> on the tropical green alga <Emphasis Type="Italic">Caulerpa taxifolia</Emphasis>

The feeding behaviour of the shelled sacoglossan Oxynoe olivacea was investigated to better understand the role and importance of this species in influencing encroachments of the alien alga Caulerpa taxifolia in the Mediterranean sea. We tested whether this slug preferred, as preliminary field observations suggested, an aggregative feeding behaviour and which part of the algal thallus, phylloid vs rhizoid, it preferred. Results showed that O. olivacea fed in groups and actively selected phylloid. This outcome poses important questions regarding the possibility that this species, fragmenting the alga thallus, could enhance dispersion and regeneration of C. taxifolia.     

Fine structure of the phylogenetically important marine algaRhodogorgon carriebowensis (Rhodophyta,Batrachospermales?)

Summary The fine structure of the recently described red algaRhodogorgon carriebowensis J. Norris et Bucher was studied by light microscopy and scanning and transmission electron microscopy to aid in the ordinal placement of this unusual alga. Most significant in this context were findings that pit plugs had two-layered plug caps, the outer layer of which formed a large dome and was composed of glycoprotein. Cap membranes appeared to be absent. Medullary cells were remarkable in having extremely thick, layered cell walls, whose inward deposition left little room for cytoplasm. Medullary filaments branched little except near the base of the cortex. The assimilatory filaments that made up most of the cortex contained almost all the pigmentation and starch reserves of the thallus. These filaments terminated in either slender apical cells with smooth cell walls or bulbous apical cells bearing spinulose projections. Two types of elongated cells were found in the cortex, those with calcified cell walls and those surrounded by multiple, spreading layers of wall material. Neither cell type was pigmented. The latter type sometimes supported normal assimilatory cells.The hypothesis is proposed thatRhodogorgon is a descendant of the marine progenitors of the Batrachospermales and thus is a member of this order.Abbreviations DIC differential interference contrast - PAS periodic acid Schiff - PTA-CA phosphotungstic acid-chromic acid     

The amino acid transport systems of the autotrophically grown green alga Chlorella

The kinetic analysis of l-amino acid uptake by the green alga Chlorella revealed at least seven different uptake systems to be present in cells grown autotrophically with nitrate as nitrogen source. There is a ‘general system’ which transports most neutral and acidic amino acids, a system for short-chain neutral amino acids including proline, a system for basic amino acids including histidine, a special system for acidic amino acids, and specific systems for methionine, glutamine and threonine. The ‘general system’ is possibly the same as that which can be stimulated by incubation of cells in glucose plus ammonium (Sauer, N. (1984) Planta 161, 425–431). The incubation of Chlorella in glucose induces the increased synthesis of six amino acid uptake systems, namely the above-mentioned system for short-chain neutral amino acids, a threonine system, a methionine system, and a glutamine system. These results indicate that the uptake of l-amino acids by the green alga Chlorella is as complex as in other free-living organisms such as bacteria or yeast. The small number of amino acid uptake systems found in cells of higher plants, i.e. two or three, seems therefore to be a consequence of integration of the cells in a tissue supplying a relatively constant environment, and not a consequence of autotrophic growth on mineral carbon and mineral nitrogen.     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

N-15 in vivo NMR spectroscopic investigation of nitrogen deprived cell suspensions of the green alga Chlorella fusca

The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K¹⁵NO₃ after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected. Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids. The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo NMR spectroscopy can be applied to the investigation of N metabolism of the cells.     

Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales,Chlorophyta)

Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 10⁵ cells ml⁻¹ before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Molecular cloning of yeast cytochrome C-like polypeptide expressed in human lung carcinoma: An antigen recogizable by lung cancer-specific human monoclonal antibody

Summary We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c fromCandida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. Thein vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297–475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.     

The distribution of colonies of the bryozoan Antarctothoa bougainvillei on the red alga Hymenena laciniata

The cheilostome bryozoan Antarctothoa bougainvillei (d’Orbigny) is the most frequent epibiont on the ribbon-like red alga Hymenena laciniata (Hooker f. & Harvey) Kylin in San Sebastián Bay (Tierra del Fuego, Argentina). Twenty-one thalli and 1,484 colonies were examined to analyse the relationship between both species. In most cases, number and area of colonies did not differ significantly at both sides of the thallus. Ancestrulae (i.e., founder zooids originating colonies by asexual budding) were mostly oriented facing the algal growing edge. Colonies were more frequent on central than on marginal zones of the thalli. The population of A. bougainvillei was mainly composed of very small colonies (<10 mm²). Larger colonies predominated and intraspecific competition was more intense near the basal portions of the thalli. Fecundity (number of ovicells) increased at a significantly higher rate in colonies with margins obstructed by conspecific neighbours than in free-growing colonies. Colonies were significantly larger on somatic than on reproductive algal tissues. As total and reproductive surfaces covered by colonies of A. bougainvillei were on average very low (4.43% and 0.53%, respectively), this epibiont is not supposed to produce a negative effect on H. laciniata. Handling editor: T. P. Crowe     

A unique pattern of F-actin organization supports cytokinesis in vacuolated cells of Macrocystis pyrifera (Phaeophyceae) gametophytes

Summary. The organization of actin filaments and their role in cytokinesis was studied in regenerating protoplasts and thallus cells of gametophytes of the brown alga Macrocystis pyrifera. Before the onset of cytokinesis, a ring of actin filaments appeared on the putative cytokinetic plane just under the plasmalemma. Light and electron microscopy of cytokinetic cells revealed that large vacuoles occupy the space between the daughter nuclei, which very often are eccentrically positioned at the cell cortex. By the progress of cytokinesis, actin filament bundles emanating from the cytokinetic ring tend to form an actin plate that enters cytoplasmic pockets in which the cytokinetic diaphragm develops. The mechanism of this cytokinetic pattern that has not been reported so far for brown algae is discussed. Correspondence and reprints: Department of Botany, Faculty of Biology, University of Athens, Athens 157 84, Greece.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

Cloning and expression of a gene coding for the major light-harvesting chlorophyll a/b protein of photosystem II in the green alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Genes encoding proteins of the major light-harvesting complex of photosystem II (LHCII) in higher plants are well studied. However, little is known about the corresponding genes in the green alga Dunaliella salina, although this knowledge might provide valuable information about the respective roles of each LHCII protein at the molecular level under extreme environmental conditions. Here, we describe an additional LhcII gene from D. salina. An LhcII cDNA cloned by screening a D. salina cDNA library contains an open reading frame encoding a protein of 261 amino acids with a calculated molecular mass of 27.8 kDa. The deduced amino acid sequence shows high homology with other LHCII proteins. Genomic DNA—obtained by PCR using a specific primer set corresponding to the 5′ and 3′ untranslated regions—was used to determine the intron-exon structure. Short-term changes in mRNA levels after a shift from low-light to high-light or dark conditions were analyzed by real-time quantitative PCR, and indicated that this gene expresses different mRNA levels under different light conditions.     

Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum: identification and sequencing of a trpGDC cluster

Summary Screening the tryptophan (Trp)-dependent indole-3-acetic acid (IAA) production of different Azospirillum species revealed that A. irakense KA3 released 10 times less IAA into the medium than A. brasilense Sp7. A cosmid library of strain Sp7 was transferred into A. irakense KA3 with the aim of characterizing genes involved in IAA biosynthesis. Trp-dependent IAA production was increased in two transconjugants which both contained an identical 18.5 kb HindIII fragment from Sp7. After Tn5 mutagenesis, cosmids carrying Tn5 insertions at 36 different positions of the 18.5 kb fragment were isolated and transferred into strain KA3. IAA production by the recipient strains was screened by HPLC. The Tn5 insertions of 4 clones with decreased IAA production were mapped on a 2 kb Sall — SphI fragment. Recombination of Tn5 insertions at this locus into the genome of strain Sp7 led to Trp auxotrophic mutants. A 5.2 kb EcoRI — SalI fragment including the kb SalI — SphI fragment was sequenced and six open reading frames were identified. Three of them were clustered and their deduced amino acid sequences showed significant similarity to TrpG, TrpD and TrpC, which are enzymes involved in tryptophan biosynthesis. One of the remaining open reading frames probably encodes an acetyltransferase. The region responsible for the enhanced Trp-dependent IAA production in strain KA3 corresponded to trpD, coding for the phosphoribosyl anthranilate transferase.     

Fine structure of the vegetative thallus of the lichen,Peltigera polydactyla

Summary An investigation was made of the vegetative thallus of the lichen Peltigera polydactyla. Using a modified embedding technique it was shown that the ultrastructure of the mycobiont was not radically different from that of nonlichenized Discomycetes, and that the ultrastructure of the phycobiont was like that of the blue-green alga Nostoc. In addition to what were considered healthy blue-green algal cells other cells were present which obviously were breaking down. Well defined heterocysts were also present. No haustoria were found in the thallus.     

Meristem clusters in cortical layer of thallus cells of the cultured red alga Palmaria palmata

For the first time somatic spotlike formations of 1.5 mm in diameter and height up to 0.5 mm were revealed in thalli of the red alga Palmaria palmata reared in an aerated stirred culture. It was determined that some cortical cells of the thallus are able to divide in the periclinal and anticlinal directions forming spots. Deep freezing and subsequent thawing of thalli showed that the cortical cells of spots were meristematic cells. In certain conditions they enabled the formation of prolifications, which germinated plantlets. These clusters of meristem tissue in the cortical layer of cells of the thallus of P. palmata, which were formed in the culture as in nature, function as growth cells facilitating growth of thalli in thick and natural “planting material” formed upon thalli fragmenting after their freezing in the winter season.     

Rapid isolation and sequence analysis of the beta-tubulin gene from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

Beta-tubulin, one of the cytoskeletal proteins, has been highly conserved throughout the evolution of eukaryotes. Degenerate PCR and inverse PCR (iPCR) were used to isolate the full-length beta-tubulin gene and its 5′ and 3′-flanking regions (2799bp) from the marine red alga Porphyra yezoensis. This gene, designated as TubB1, is devoid of introns. The canonical cis-acting elements such as TATA box, CAAT box and polyA signal AAUAAA are not found in flanking sequences, but another putative polyA signal CAYTG is found downstream of the stop codon. Comparison of the deduced 458 amino acid sequences shows higher similarity to the Protoflorideophycidae Cyanidioschyzon merolae (82%) than to the red alga Chondrus crispus (79%). Codon bias indicates strong expression of TubB1. Phylogenetic analysis suggests that the beta-tubulin of P. yezoensis and C. merola go together with fungi and not with green plants. These nucleotide sequence data have been deposited in the DDB/EMBL/Genbank databases under the accession number AY221630.     

Studies on the evolution of auxin carriers and phytotropin receptors: Transmembrane auxin transport in unicellular and multicellular Chlorophyta

The characteristics of transmembrane transport of ¹⁴C-labelled indol-3yl-acetic acid ([1-¹⁴C]IAA) were compared in Chlorella vulgaris Beij., a simple unicellular green alga, and in Chara vulgaris L., a branched, multicellular green alga exhibiting axial polarity and a high degree of cell and organ specialization. In Chara thallus cells, three distinguishable trans-plasmamembrane fluxes contributed to the net uptake of [1-¹⁴C]-IAA from an external solution, viz.: a non-mediated, pH-sensitive influx of undissociated IAA (IAAH); a saturable influx of IAA; and a saturable efflux of IAA. Both saturable fluxes were competitively inhibited by unlabelled IAA. Association of [³H]IAA with microsomal preparations from Chara thallus tissue was competitively inhibited by unlabelled IAA. Results indicated that up-take carriers occurred in the membranes at a much higher density than efflux carriers. The efflux component of IAA net uptake by Chara was not affected by several phytotropins (N-1-naphthylphthalmic acid, NPA; 2-(1-pyrenoyl)benzoic acid; and 5-(2-carboxyphenyl)-3-phenylpyrazole), which are potent non-competitive inhibitors of specific auxin-efflux carriers in more advanced plant groups, and no evidence was found for a specific association of [³H]NPA with Chara microsomal preparations. It was concluded that Chara lacked phytotropin receptors. Net uptake of [1-¹⁴C]IAA also was unaffected by 2,3,5-triiodobenzoic acid except at concentrations ( 10^–1 mol · m^–3) high enough to depress cytoplasmic pH (determined by uptake of 5,5-dimethyloxazolidine-2,4-dione). Chlorella cells accumulated [1-¹⁴C]IAA from an external solution by pH-sensitive diffusion of IAA across the plasma membrane and anion (IAA^–) trapping, but no evidence was found in Chlorella for the occurrence of IAA carriers. These results indicate that carrier systems capable of mediating the transmembrane transport of auxins appeared at a very early stage in the evolution of green plants, possibly in association with the origin of a differentiated, multicellular plant body. Phytotropin receptors evolved independently of the carriers.Abbreviations CPP 5-(2-carboxyphenyl)-3-phenylpyrazole - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid We thank the Nuffield Foundation for the award of an Undergraduate Research Bursary to J.E.D.-F., Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia for generous gifts of phytotropins, and Mrs. R.P. Bell for technical support.     

Changes in protein profile and amino acids in <Emphasis Type="Italic">Cladophora vagabunda</Emphasis> (Chlorophyceae) in response to salinity stress

The aim of the present study was to detect organic substances functioning as osmoticants that are used by the intertidal alga, Cladophora vagabunda (L.) Hoek (Chlorophyceae), to adapt to a wide range of salinity. The major constituents of the amino acid pool were aspartate, glutamate, glycine, valine, lysine, histidine, arginine, and proline. There were concomitant increases in the acidic amino acids: aspartate and the glutamate and the basic amino acids: lysine, histidine and arginine in response to salinity stress. The appearance of proline at hypersalinity alone showed that it acts as an osmoticant. As salinity increased, there was a progressive shift in the electrophoretic pattern of protein bands. New peptide bands appeared under hyposalinity (10‰) and hypersalinity (65‰) stress conditions in addition to the usual bands which appeared in the control (35‰). Glycine betaine, which has been considered a novel organic osmolyte in a number of organisms, has also been observed in C. vagabunda in response to salinity stress. The synthesis of the compatible solute glycine betaine and the amino acid proline with increasing salinity illustrates the contention that marine algae establish an osmotic equilibrium primarily by the synthesis of organic compounds. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.