Tatyana A. Klochkova

PURIFICATION AND CHARACTERIZATION OF A LECTIN, BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) 1

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N‐acetyl‐d‐glucosamine and N‐acetyl‐d‐galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate‐labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N‐acetyl‐d‐galactosamine and N‐acetyl‐d‐glucosamine in the crude extract. One‐step column purification using N‐acetyl‐d‐glucosamine‐agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross‐linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The N‐terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”

Olpidiopsis sp., an oomycete from Madagascar that infects Bostrychia and other red algae : Host species susceptibility

Olpidiopsis sp. (Oomycota) was cultured with its original host Bostrychia moritziana (Sonder ex Kützing) J. Agardh from Madagascar. Bean‐shaped zoospores with two heterokont flagella attached to the host cell wall surface and in 2 days host cells began collapsing and one or more syncytia developed in each infected cell. Zoospores were cleaved and an exit tube with a small plug was formed. Complete development and zoospore discharge occurred in 3 days. Infection occurred in cells of polysiphonous branches, monosiphonous branches, rhizoids and reproductive stichidia. Dead cells of plants treated with microwave were not infected. Susceptibility was variable in other Bostrychia species from different countries. Bostrychia moritziana (Sonder ex Kützing) J. Agardh, and Bostrychia radicans (Montagne) Montagne from Madagascar were susceptible but one Bostrychia tenella (J. V. Lamouroux) J. Agardh isolate from Madagascar was susceptible and two were not. B. radicosa (Itono) J. A. West, G. C. Zuccarello et M. Hommersand isolates from Madagascar, Thailand, Australia and New Caledonia were susceptible but an isolate from Malaysia was not. B. radicans isolates from Mexico and Brazil were non‐susceptible as were Bostrychia flagellifera Post, Bostrychia harveyi Montagne, Bostrychia montagnei Harvey, Bostrychia simpliciuscula Harvey ex J. Agardh, Bostrychia tenuissima R. J. King et Puttock, Stictosiphonia intricata(Bory de Saint‐Vincent) P. C. Silva, Stictosiphonia kelanensis (Grunow) R. J. King et Puttock and Stictosiphonia tangatensis (Post) R. J. King et Puttock, Lophosiphonia sp., Neosiphonia sp. and Polysiphonia spp. isolates were also non‐susceptible. Many non‐susceptible strains showed initial cell‐collapse followed by rapid wound‐repair cell formation without syncytia or sporangia developing. Caloglossa leprieurii (Montagne) G. Martens from Madagascar showed cell‐collapse and wound‐repair in periaxial cells, but wing cells died and became purple without wound‐repair. Caloglossa ogasawaraensis Okamura and Caloglossa postiae M. Kamiya et R. J. King had no symptoms of infection. Dasysiphonia chejuensis I. K. Lee et J. A. West was not infected. Surprisingly, the conchocelis phase but not the blade phase of Porphyra pulchella J. A.West, G. C. Zuccarello and Porphyra suborbiculata Kjellman was infected. The conchocelis of Porphyra tenera Kjellman and Porphyra linearis Greville were infected but no blade stages were tested. Porphyra miniata (C. Agardh) C. Agardh and Porphyra dentata Kjellman conchocelis were not infected. Bangia atropurpurea (Roth) C. Agardh gametophyte filaments were not infected. Other red, brown and green algae were not infected. Time lapse videomicroscopy of development and spore release was done.

MOLECULAR CHARACTERIZATION OF THE LECTIN, BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)1

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5′ untranslated region (UTR) and 201 bp of 3′ UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C‐terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.

Biology of a terrestrial green alga, Chlorococcum sp. (Chlorococcales, Chlorophyta), collected from the Miruksazi stupa in Korea

T.A. Klochkova, S.-H. Kang, G.Y. Cho, C.M. Pueschel, J.A. West and G.H. Kim. 2006. Biology of a terrestrial green alga, Chlorococcum sp. (Chlorococcales, Chlorophyta), collected from the Miruksazi stupa in Korea. Phycologia 45: 349–358. DOI: 10.2216/04-58.1 A terrestrial chlorophyte, Chlorococcum sp., was isolated from the stone walls of Miruksazi stupa, which is a national treasure of Korea. The alga was one of the dominant organisms contributing to biodeterioration of the monument and it grew extensively on the walls of the inner room of the stupa, which had been sealed for more than 5 yr before we started this experiment. Chlorococcum survived in darkness during that time as dormant, warty, thick-walled spores. The resting spores revived in freshwater medium and released numerous unicellular progeny, which were isolated into a unialgal culture. The isolate was subjected to 18S rDNA phylogenetic analysis as well as ultrastructure and life cycle studies. In addition, the effect of salinity stress was investigated using sterile enriched seawater as a medium. Chlorococcum sp. grew in seawater culture medium for more than 5 mo and reproduced by aplanospores.

Olpidiopsis bostrychiae sp. nov.: an endoparasitic oomycete that infects Bostrychia and other red algae (Rhodophyta)

Sekimoto S., Klochkova T.A., West J.A., Beakes G.W. and Honda D. 2009. Olpidiopsis bostrychiae sp. nov.: an endoparasitic oomycete that infects Bostrychia and other red algae (Rhodophyta). Phycologia 48: 460–472. DOI: 10.2216/08-11.1. A holocarpic, oomycete endoparasite of the marine red alga Bostrychia moritziana collected in Madagascar and identified as Olpidiopsis sp. did not conform to morphological descriptions of any species of Olpidiopsis and had a host range that differed from that of known Olpidiopsis pathogens of red algae. We further tested the host range of this parasite and studied it by transmission electron microscopy and molecular means. It differs from other Olpidiopsis species, and we have named it O. bostrychiae. Molecular phylogenies inferred from SSU rRNA gene and COII amino acid sequences showed that O. bostrychiae branched before the main saprolegnialean and peronosporalean lineages within the monophyletic oomycetes. In the SSU rRNA gene tree, O. bostrychiae formed a clade with O. porphyrae, which infects Bangia and Porphyra spp. The two Olpidiopsis species were genetically and ultrastructurally distinct, supporting our decision to name an additional Olpidiopsis species that is parasitic on red algae.

THE UTILITY OF PROTEOMICS IN ALGAL TAXONOMY: BOSTRYCHIA RADICANS/B. MORITZIANA (RHODOMELACEAE, RHODOPHYTA) AS A MODEL STUDY1

A comparison of the proteome of eight genetically well‐characterized isolates of the Bostrychia radicans (Mont.) Mont./B. moritziana (Sond. ex Kütz.) J. Agardh species complex was undertaken to establish if genetic relationships among them can be determined using proteome data. Genetic distances were calculated on the basis of common and distinct spots in two‐dimensional gel electrophoresis (2‐DE). Proteomes of the male and female plants of each population were compared to analyze the range of genetic difference within an isolate. Haploid male and female plants of the same species had 3.7%–7.1% sex‐specific proteins. The degree of similarity of the proteome was consistent with previous DNA sequence data and sexual compatibility studies between the isolates. Two sexually compatible isolates from Venezuela showed a pair‐wise distance ranging from 0.14 to 0.21. The isolates from Mexico and Venezuela, which were partially compatible, showed a maximum pair‐wise distance of 0.26. A high level of genetic difference was found among isolates that were sexually incompatible. The isolate from Brazil was reproductively isolated from the Mexico and Venezuela isolates and showed a maximum pair‐wise distance of 0.65 and 0.58, respectively. Comparative proteomics may be helpful for studying genetic distances among algal samples, if intraisolate variation (gene expression) can be minimized or tested.

Possible surface carbohydrates involved in signaling during conjugation process in Zygnema cruciatum monitored with fluorescein isothiocyanate-lectins (Zygnemataceae, Chlorophyta)

The changes of cell surface carbohydrates were examined with FITC (fluorescein isothiocyanate)‐labeled lectins during the conjugation process of the green alga Zygnema cruciatum. The Ulex europaeus agglutinin (UEA)‐specific materials were detected consistently on the surface of vegetative cells, but were absent on the surface of protruding papillae or conjugation tube. The tips of male and female papillae were labeled with soybean agglutinin (SBA) and peanut agglutinin (PNA) during conjugation. The SBA‐ and PNA‐specific materials appeared first at the tip of male papillae and began to accumulate on the surface of female papillae. No labeling of these lectins was detected on the surface of vegetative filaments throughout the conjugation process. FITC‐ConA (Concanavalin A) and FITC‐RCA (Ricinus communis agglutinin) did not label the vegetative filaments of Z. cruciatum, but a trace labeling of these lectins was observed on the surface of some swollen papillae occasionally. Blocking experiments with various lectins showed that these SBA‐ and PNA‐specific glycoconjugates might be involved in the signaling between male and female papillae.

PURIFICATION OF A SEX-SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.

Isolation and Characterization of a Sex-Specific Lectin in a Marine Red Alga, Aglaothamnion oosumiense Itono

ABSTRACT In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-d-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding.

Experimental Hybridization between Some Marine Coenocytic Green Algae Using Protoplasms Extruded in vitro

Some marine coenocytic green algae could form protoplasts from the extruded protoplasm in seawater. The dissociated cell components of the coenocytic protoplasm could be reunited into live cells and, hence, the formation of new species by mixing protoplasms from different coenocytic cells has been predicted. Our results showed that an incompatibility barrier was present during protoplast formation in coenocytic algae to exclude foreign inorganic particles or alien cell components. No inorganic particles or alien cell components were incorporated into protoplast formed spontaneously in seawater. Even when the inorganic particles or alien cell and/or cell component were incorporated into protoplast in some experimental condition, they were expelled from the protoplast or degenerated within several days. A species-specific cytotoxicity was observed during protoplast hybridization between the protoplasms of Bryopsis spp. and Microdictyon umbilicatum. The cell sap of M. umbilicatum could destroy the cell components of Bryopsis spp., but had no effect on Chaetomorpha moniligera. Species C. moniligera and Bryopsis did not affect protoplast generation of either species. The wound-induced protoplast formation in vitro might have evolved in some coenocytic algae as a dispersal method, and the incompatibility barrier to alien particles or cell and/or cell component could serve as a protective mechanism for successful propagation.