THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. I. DEVELOPMENT AND CHARACTERISTICS OF A BINDING ASSAY1

To explore the molecular basis of egg-sperm recognition in the brown alga , Fucus serratus L., we developed an in vitro binding assay involving egg plasma membrane vesicles (PMVs) and proteins contained in a KCl extract of sperm. Binding between the two components was measured using biotinylated PMVs followed by the addition of streptavidin conjugated to alkaline phosphatase and the appropriate substrate. Biotinylation did not affect the ability of egg PMVs to inhibit fertilization in a species-preferential manner. Binding of labeled egg PMVs to the sperm KCl extract was saturable and competable with unlabeled PMVs but was not species-specific. Protease treatment of the KCl extract abolished binding, whereas periodate had no effect, suggesting that sperm protein rather than carbohydrate was involved. Preincubation of the sperm extract with sulfated polysaccharides (e.g. fucoidan and ascophyllan) inhibited binding of egg plasma membranes. Sulfation seems to be important for this effect since desulfated fucoidan was far less effective at blocking binding. Polysaccharides which inhibited binding also inhibited fertilization. Overall, the results indicate that at least some aspects of binding between Fucus sperm and eggs are mediated by a protein(s) derived from sperm which recognizes sulfated glycoconjugates on the egg plasma membrane .     

INHIBITION OF FERTILIZATION IN FUCUS (PHAEOPHYCEAE) BY A MONOCLONAL ANTIBODY THAT BINDS TO DOMAINS ON THE EGG CELL SURFACE1

Fertilization in the brown alga Fucus serratus L. involves interactions between sperm and eggs and in species-specific. Recent results with monoclonal antibodies(MAbs FS4 and FS5) that recognize glycoproteins on the Fucus egg cell surface have shown that different sets of glycoproteins are organized into distinct domains. Thus, regions on the egg surface are FS4⁺FS5^? and FS4^?FS5⁺, and some smaller regions are FS4⁺FS⁺. To determine functional differences between these domains, MAbs FS4 and FS5 were included in a fertilization assay, Purified FS5 antibody, which is an immunoglobulin class M (IgM), inhibited fertilization, whereas FS4 IgM had no effect, Fragment antigen-binding fragment, which suggests that the effects were not due to steric hindrance by large IgM molecules or to cross-linking of receptors. FS5 inhibited fertilization in both F. serratus and F. vesiculosus; therefore, its effect was not species-specific, Thus we show that there is functional heterogeneity of the Fucus egg cell surface and compare our results to previous reports on inhibitory effects of lectins, Overall, the evidence suggests that there may be two levels of interaction between gametes in Fucus involoving nonspecific and species-specific binding.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

PREMATURE CHROMOSOME CONDENSATION OF THE KARYOGAMY-BLOCKED SPERM PRONUCLEUS IN THE FERTILIZATION OF FUCUS DISTICHUS (FUCALES,PHAEOPHYCEAE)1

Mitosis of egg and sperm pronuclei of Fucus distichus subsp. evanescens (C. Agardh)Powell was examined by fluorescence and electron microscopy when migration of the sperm pronucleus and, as a result, karyogamy were blocked by colchicine treatment after plasmogamy. Chromosome condensation was obsewed in both pronuclei Microspectrophotometric studies after staining the nuclei with mithramycin A clearly showed that DNA synthesis ocurred in the egg pronucleus but not in the sperm pronucleus. This means that chromosomes condensed prematurely in the sperm pronucleus (premature chromosome condensation). In some cases, the egg chromosomes became arranged on a metaphase plate, whereas the sperm chromosomes lay scattered near the egg pronucleus. Immuno fluorescence microscopy using anti-β-tubulin antibody confirmed that a normal spindle was formed at the egg pronucleus. A pair of centrioles existed at the two poles of this spindle. The sperm nuclear membrane disappeared, and microtubules radiated to the sperm chromosomes from one pole of the egg spindle.     

EVOLUTION OF MACROCYSTIS SPP. (PHAEOPHYCEAE) AS DETERMINED BY ITS1 AND ITS2 SEQUENCES1,

Macrocystis (Lessoniaceae) displays an antitropical distribution, occurring in temperate subtidal regions along western North America in the northern hemisphere and throughout the southern hemisphere. We used the noncoding rDNA internal transcribed spacer regions (ITS1 and ITS2) to examine relatedness among (1) Macrocystis and several genera of Laminariales, (2) four species of Macrocystis ( M. integrifolia Bory from the northern hemisphere, M. angustifolia Bory and M. laevis Hay from the southern hemisphere, and M. pyrifera [L.] C. Ag. from both hemispheres), and (3) multiple clones of several individuals. Of the taxa included in our phylogenetic analysis, the elk kelp, Pelagophycus porra (Lem.) Setch., was the sister taxon to Macrocystis spp. Macrocystis individuals from the southern hemisphere (representing three species) formed a strongly to moderately supported clade, respectively, when the ITS1 and ITS2 sequences were analyzed separately. No distinction was detected between the two species in the northern hemisphere. Thus, Macrocystis may be a monospecific genus ( M. pyrifera ). A northern-hemisphere-to-southern-hemisphere pattern of dispersal was inferred, because northern-hemisphere individuals were more diverse and displayed paraphyletic clades, whereas southern-hemisphere individuals were less diverse and formed a monophyletic clade. High intraindividual variation in ITS1 sequences was observed in one individual from Santa Catalina Island (CA), suggesting very recent and rapid mixing of genotypes from areas to the north and Baja California (Mexico) or introgressive hybridization with Pelagophycus.     

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)1

Closterium strains obtained from Japan ( NIES-64 and -65 ) and Nepal ( NIES-67 and -68 ) have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES-64 and -65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS-67 or -68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES-68, called the protoplast-release-inducing protein ( PR-IP ), had no effect on the release of protoplasts from cells of strains NIES-64 or -65. When the media obtained from the culture of NIES-64 and -65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunits of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not structurally similar to PR-IP. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IP inducer ) which is released by NIES-68 cells. In contrast, no protoplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES-64/ NIES-65 and strains NIES-67 /NIES-68 differ, and we propose a possible mechanism of sexual isolation between these biological species .     

A CLADISTIC ANALYSIS OF THE EVOLUTION AND BIOGEOGRAPHY OF DURVILLAEA (PHAEOPHYTA)1

The genus Durvillaea currently has four recognized species found along many exposed, rocky coastlines of the temperate to sub-Antarctic regions in the Southern Hemisphere. We propose that the current species distributions are related primarily to vicariance events and subsequent speciation associated with the breakup of Gondwana between 40 and 100 Ma. From an ancestral species, a stipitate species developed in the Tasman basin, with separation and speciation resulting in the D. potatorum/ D. willana complex in southeastern Australia and New Zealand. A second line of evolution led to D. chathamensis and D. antarctica characterized by a honeycombed medulla. The extensive distribution of D. antarctica throughout the Southern Hemisphere is related to both vicariance and dispersal events. The status of D. chathamensis as a species distinct from D. antarctica is questioned. The affinities of an as yet undescribed taxon from the Antipodes Islands are thought to be with the D. potatorum complex but require further study before they can be defined more precisely .     

CLONING OF A cDNA ENCODING A 66-kDa Ca2+-DEPENDENT PROTEIN KINASE (CDPK) FROM DUNALIELLA TERTIOLECTA (CHLOROPHYTA)

A cDNA clone encoding a Ca²+-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1 , a CDPK from the green alga, Chlamydomonas eugamentos Moewus . The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca²+-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca²+ concentration in vivo .     

A MOLECULAR PHYLOGENY OF THE GELIDIALES (RHODOPHYTA) BASED ON ANALYSIS OF PLASTID rbcL NUCLEOTIDE SEQUENCES1

Parsimony analyses of rbcL nucleotide. sequences were used to develop hypotheses of relationships among taxa in the taxonomically difficult order Celidiales including species from seven currently recognized genera: Capreolia, Gelidiella, Gelidium, Onikusa, Pterocladia, Ptilophora, and Suhria. Nucleotide. sequences of rbcL from red algae are variable and provide a large number of informative characters for phylogenetic analysis, yet the absence of insertion/deletion mutations allows for the unambiguous alignment of sequences. Species were resolved into 10 well-.supported major clades representing genera and species complexes. The topological positions of these 10 clades within trees are also well supported and indicate that Gelidium and Pterocladia as currently circumscribed are not monophyletic. These results call for a revision of the classification of the Gelidiales .     

ISOLATION OF A SULFOQUINOVOSYL MONOACYLGLYCEROL FROM BRYOPSIS SP. (CHLOROPHYTA): IDENTIFICATION OF A FACTOR CAUSING A POSSIBLE SPECIES-SPECIFIC ECDYSIS RESPONSE IN GAMBIERDISCUS TOXICUS (DINOPHYCEAE)1,

A bioactive compound that induced ecdysis (thecal loss) in Gambierdiscus toxicus Adachi et Fukuyo (Dinophyceae) cultures was isolated from a host macroalga, Bryopsis sp. (Chlorophyta). The ecdysis factor was identified by spectroscopic methods as 1- O -palmitoyl-3- O -(6'-sulfo-α- d -quinovopyranosyl)- sn -glycerol (PSQG). From our results, PSQG induced ecdysis at a high frequency and appeared not to inhibit the growth of G. toxicus cultures. The induction of ecdysis followed a dose-dependent saturation curve from 4 to 8 μM PSQG. To determine specificity of PSQG, the effects of palmitoyl- l -α-lysophosphatidylcholine (PLPC) were observed. Because PLPC contains a lipophilic palmitoyl moiety and hydrophilic phosphatidyl choline group, the compound possesses a detergent-like amphiphathic property similar to PSQG. Our results demonstrate that PLPC induced ecdysis at a high frequency in G. toxicus cultures and generated a similar dose–response curve as PSQG. The ecdysis activity observed in PSQG and PLPC may correlate with the detergent-like amphiphathic property of both compounds. Although PLPC induced a similar ecdysis response as PSQG, PLPC appeared to inhibit the growth of G. toxicus cultures. Preliminary results on the effects of PSQG on the dinoflagellates Prorocentrum lima (Ehrenberg) Dodge and Coolia monotis Meunier did not parallel the results observed in G. toxicus. This study demonstrated the existence of a factor from Bryopsis sp. that elicited a possible species-specific ecdysis response in G. toxicus cultures. This is the first report of a compound that induced ecdysis in G. toxicus or in any dinoflagellate.     

V- AND P-TYPE Ca2+-STIMULATED ATPases IN A CALCIFYING STRAIN OF PLEUROCHRYSIS SP. (HAPTOPHYCEAE)

Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane-bound Ca²⁺-stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca²⁺-stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca²⁺-stimulated ATPase. The Ca²⁺-stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP-dependent H⁺ movement, but not ⁴⁵Ca²⁺ transport, across the coccolith vesicle was demonstrated. The Ca²⁺-stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP-dependent, vanadate-sensitive efflux of ⁴⁵Ca²⁺ was demonstrated for microsomal material derived from gradient-isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross-reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross-react. These findings show that a V-type Ca²⁺-stimulated ATPase is located on the coccolith vesicle membrane and a P-type Ca²⁺-stimulated ATPase is located on the plasma membrane.     

A HYPOTHESIS FOR IMPORT OF THE NUCLEAR‐ENCODED PsaE PROTEIN OF PAULINELLA CHROMATOPHORA (CERCOZOA,RHIZARIA) INTO ITS CYANOBACTERIAL ENDOSYMBIONTS/PLASTIDS VIA THE ENDOMEMBRANE SYSTEM1

The cyanobacterial endosymbionts of Paulinella chromatophora can shed new light on the process of plastid acquisition. Their genome is devoid of many essential genes, suggesting gene transfer to the host nucleus and protein import back into the endosymbionts/plastids. Strong evidence for such gene transfer is provided by the psaE gene, which encodes a PSI component that was efficiently transferred to the Paulinella nucleus. It remains unclear, however, how this protein is imported into the endosymbionts/plastids. We reanalyzed the sequence of Paulinella psaE and identified four potential non‐AUG translation initiation codons upstream of the previously proposed start codon. Interestingly, the longest polypeptide, starting from the first UUG, contains a clearly identifiable signal peptide with very high (90%) predictability. We also found several downstream hairpin structures that could enhance translation initiation from the alternative codon. These results strongly suggest that the PsaE protein is targeted to the outer membrane of Paulinella endosymbionts/plastids via the endomembrane system. On the basis of presence of respective bacterial homologs in the Paulinella endosymbiont/plastid genome, we discuss further trafficking of PsaE through the peptidoglycan wall and the inner envelope membrane. It is possible that other nuclear‐encoded proteins of P. chromatophora also carry signal peptides, but, alternatively, some may be equipped with transit peptides. If this is true, Paulinella endosymbionts/plastids would possess two distinct targeting systems, one cotranslational and the second posttranslational, as has been found in higher plant plastids. Considering the endomembrane system‐mediated import pathway, we also discuss homology of the membranes surrounding Paulinella endosymbionts/plastids.     

INFERRING TAXONOMIC POSITIONS AND TESTING GENUS LEVEL ASSIGNMENTS IN COCCOID GREEN LICHEN ALGAE: A PHYLOGENETIC ANALYSIS OF 18S RIBOSOMAL RNA SEQUENCES FROM DICTYOCHLOROPSIS RETICULATA AND FROM MEMBERS OF THE GENUS MYRMECIA (CHLOROPHYTA, TREBOUXIOPHYCEAE CL. NOV.)1

Complete nuclear-encoded small-subunit ribosomal RNA (18S rRNA) coding sequences were determined for the coccoid green algae Dictyochloropsis reticulata (Tschermak-Woess) Tschermak-Woess , Myrmecia astigmatica Vinatzer, and M. bisecta Reisigl, to investigate the taxonomic position of Dictyochloropsis Geitler and of the genus Myrmecia Printz. Phylogenies inferred from these data revealed a sister-group relationship between D. reticulata and certain coccoid green algae that lack motile stages (autosporic coccoids) within the order Microthamniales. The monophyletic origin of the Microthamniales, including autosporic coccoids previously classified in the Chlorophyceae, is clearly resolved by the rRNA sequence data. This finding. shows the considerable taxonomic breadth of that order, whose taxonomic position has been unclear so far. A new class, Trebouxiophyceae, is proposed for this group of green algae. Phylogenetic inferences from the rRNA sequences show paraphyly of the genus Myrmecia. The 18S rRNA sequence data suggest that, among taxa that share similar vegetative cell morphologies, the zoospore characters resolve better the actual genus and species boundaries. Within identical zoospore types, the rRNA data allow further resolution of taxonomic relationships. On the basis of the.se findings, I propose that the genus Friedmannia Chantanachat ± Bold be merged into Myrmecia and that only those species be left in the genus Myrmecia that are identical in particular zoospore characters (i.e. those described in detail for M. israeliensis ( Chantanachat ± Bold) comb, nov.), namely M. astigmatica, M. biatorellae (Tschermak-Woess ± Ptesst) Petersen, and M. israeliensis. Myrmecia bisecta has to be excluded from Myrmecia; its taxonomic position within the Trebouxiophyceae is unclear .