Mitosis in the coenocytic green algaBoergesenia forbesii (Harvey) Feldmann (Siphonocladales,Ulvophyceae)

Mitosis in vegetative cells of the siphonocladalean algaBoergesenia forbesii (Harvey) Feldmann was investigated mainly by electron microscopy. The mitotic spindle was centric and closed. The interphase nucleus contained a spherical nucleolus. The nucleolus was slightly dispersed at prophase, but nucleolar materials remained during nearly all stages of mitosis. Kinetochores were evident on chromosomes. The polar regions of nuclear envelope had no fenestrae during mitosis. Anaphase separation of the chromosomes was asynchronous. Elongation of interzonal spindle at telophase separated the two daughter nuclei widely. The ultrastructural features of mitosis inB. forbesii revealed by the present investigation are compared with those of other siphonous and siphonocladous algae in the Ulvophyceae.     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae)

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.     

Development of microsatellite markers in the green algae Enteromorpha intestinalis (Chlorophyta)

The fouling green algae Enteromorpha intestinalis is a cosmopolitan benthic species, which causes green tides in many coastal areas and is used as an indicator species for eutrophication in the Baltic Sea area. The life cycle of E. intestinalis alternates between two morphologically identical reproductive stages, a haploid gametophyte phase and a diploid sporophyte phase. However, it also reproduces through asexual propagation. The reproductive cycles of E. intestinalis in the Baltic Sea and elsewhere are largely unknown. Here we report five polymorphic microsatellite markers developed from enriched genomic libraries. The number of alleles per locus ranged from 7 to 25.     

Mitochondrial genomes of the green macroalga Ulva pertusa (Ulvophyceae,Chlorophyta): novel insights into the evolution of mitogenomes in the Ulvophyceae

To further understand the trends in the evolution of mitochondrial genomes (mitogenomes or mtDNAs) in the Ulvophyceae, the mitogenomes of two separate thalli of Ulva pertusa were sequenced. Two U. pertusa mitogenomes (Up1 and Up2) were 69,333 bp and 64,602 bp in length. These mitogenomes shared two ribosomal RNAs (rRNAs), 28 transfer RNAs (tRNAs), 29 protein‐coding genes, and 12 open reading frames. The 4.7 kb difference in size was attributed to variation in intron content and tandem repeat regions. A total of six introns were present in the smaller U. pertusa mtDNA (Up2), while the larger mtDNA (Up1) had eight. The larger mtDNA had two additional group II introns in two genes (cox1 and cox2) and tandem duplication mutations in noncoding regions. Our results showed the first case of intraspecific variation in chlorophytan mitogenomes and provided further genomic data for the undersampled Ulvophyceae.     

Parasexual fusion products in green algae: Enteromorpha and Ulvaria (Ulvales,Chlorophyta)

Enteromorpha linza and Ulvaria oxysperma in North Carolina reproduce exclusively by asexual zoospores. Calcofluor white staining indicated that newly released zoospores lack significant cellulose cell wall material, making them suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca²⁺, PEG and centrifugation. Presumptive fusion products were identified by their larger size, twin chloroplasts and eyespots, and presence of fluorescence labelled and unlabelled portions. Parasexual fusion and karyogamy were confirmed by elevated levels of nuclear DNA in fusion cell germlings. In addition, aceto-orcein staining of fusion cell products revealed a diploid chromosome complement of 2N = 20 in Enteromorpha linza. Fusion cells were isolated by killing the more numerous adjacent unfused zoospores with 2-3 min exposure to blue light (410–490 nm). Unexposed fusion cells could be readily distinguished and recovered by micropipette at the 10-day stage.Center for Marine Science Research UNC-W Contribution No. 008.     

Ethene (ethylene) production in the marine macroalga Ulva (Enteromorpha) intestinalis L. (Chlorophyta, Ulvophyceae): effect of light-stress and co-production with dimethyl sulphide

Ethene (ethylene; H₂C = CH₂) is one of a range of non-methane hydrocarbons (NMHC) that affect atmospheric chemistry and global climate. Ethene acts as a hormone in higher plants and its role in plant biochemistry, physiology and ecology has been the subject of extensive research. Ethene is also found in seawater, but despite evidence that marine microalgae and seaweeds can produce ethene directly, its production is generally attributed to photochemical breakdown of dissolved organic matter. Here we confirmed ethene production in cultured samples of the macroalga Ulva (Enteromorpha) intestinalis. Ethene levels increased substantially when samples acclimatized to low light conditions were transferred to high light, and ethene addition reduced chlorophyll levels by 30%. A range of potential inhibitors and inducers of ethene biosynthesis were tested. Evidence was found for ethene synthesis via the 1-aminocylopropane-1-acrylic acid (ACC) pathway and ACC oxidase activity was confirmed for cell-free extracts. Addition of acrylate, a potential ethene precursor in algae that contain the compatible solute dimethylsulphoniopropionate, doubled the ethene produced but no acrylate decarboxylase activity was found. Nonetheless the data support active production of ethene and we suggest ethene may play a multifaceted role in algae as it does in higher plants.     

Identification by SDS PAGE of green seaweeds ( Ulva and Enteromorpha) used in the food industry

SDS PAGE was tested as an analytical tool for the identification of fourgreen algae (Ulva rigida, U. rotundata, Enteromorphaintestinalis, E. compressa) used as food ingredients. A referencepattern composed of the bands present all year long was performed foreach species. The pattern for Ulva rotundata consists of 7 bandslocated between 69.9 and 15.5 kDa with the presence of triplicate bandsat 29.5, 26.3 and 22.9 kDa. The pattern for Ulva rigida is consistsof three bands with apparent molecular weights of 68.5, 56.4 and 44.7kDa. The Enteromorpha compressa pattern is characterised by sixbands located between 65.8 and 19.8 kDa. A double band withmolecular weights of 23.1 and 23.9 distinguished this pattern from theothers. Six bands situated between 66.4 and 19.4 kDa with a specifictriplicate band of 25.9, 23.9 and 22.5 kDa constituted the specific patternof Enteromorpha intestinalis. SDS PAGE appears to be suitable forthe identification of green seaweed foods.     

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.     

The complete mitochondrial DNA sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) highlights distinctive evolutionary trends in the chlorophyta and suggests a sister-group relationship between the Ulvophyceae and Chlorophyceae

The mitochondrial genome has undergone radical changes in both the Chlorophyta and Streptophyta, yet little is known about the dynamics of mtDNA evolution in either of these lineages. In the Chlorophyta, which comprises four of the five recognized classes of green algae (Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae), the mitochondrial genome varies from 16 to 55 kb. This genome has retained a compact gene organization and a relatively complex gene repertoire ("ancestral" pattern) in the basal lineages represented by the Trebouxiophyceae and Prasinophyceae, whereas it has been reduced in size and gene complement and tends to evolve much more rapidly at the sequence level ("reduced-derived" pattern of evolution) in the Chlorophyceae and the lineage leading to the enigmatic chlorophyte Pedinomonas. To gain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we have determined the mtDNA sequence of Pseudendoclonium akinetum. At 95,880 bp, Pseudendoclonium mtDNA is the largest green-algal mitochondrial genome sequenced to date and has the lowest gene density. These derived features are reminiscent of the "expanded" pattern exhibited by embryophyte mtDNAs, indicating that convergent evolution towards genome expansion has occurred independently in the Chlorophyta and Streptophyta. With 57 conserved genes, the gene repertoire of Pseudendoclonium mtDNA is slightly smaller than those of the prasinophyte Nephroselmis olivacea and the trebouxiophyte Prototheca wickerhamii. This ulvophyte mtDNA contains seven group I introns, four of which have homologs in green-algal mtDNAs displaying an "ancestral" or a "reduced-derived" pattern of evolution. Like its counterpart in the chlorophycean green alga Scenedesmus obliquus, it features numerous small, dispersed repeats in intergenic regions and introns. Its overall rate of sequence evolution appears to be accelerated to an intermediary level as compared with the rates observed in "ancestral" and "reduced-derived" mtDNAs. In agreement with the finding that Pseudendoclonium mtDNA exhibits features typical of both the "ancestral" and "reduced-derived" patterns of evolution, phylogenetic analyses of seven mtDNA-encoded proteins revealed a sister-group relationship between this ulvophyte and chlorophytes displaying "reduced-derived" mtDNAs.     

Novel green fluorescent protein (GFP) baculovirus expression vectors

Baculovirus expression vectors were constructed, which contained gfp as a reporter gene. Substitutions in the amino acid sequences were carried out to produce two mutant forms of GFP. One of these mutants produces blue color when excited by ultraviolet (UV) light. The other mutant produces a yellow color that can be visualized in regular daylight. The gene of interest cloned in-frame with the gfp open reading frame allows visualization of the produced GFP-fusion protein using UV light. The presence of an in-frame 6 x His tag between the gfp cDNA and the multiple cloning site allows purification of the fusion protein on a Ni-NTA (nickel-nitrilo-triacetic acid) agarose matrix.     

Preparation of protoplasts from the green alga Enteromorpha intestinalis (L.) Link

Protoplasts have been obtained from vegetative thallus of the green seaweed Enteromorpha following enzymic digestion with driselase and pectinase. The viability of purified protoplast fractions was assessed by staining and measurements of O₂ uptake and evolution.Abbreviations MES 2-(N-morpholino) ethanesulphonic acid - TES N-tris(hydoxymethyl) methyl-2 aminoethanesulphonic acid     

An appraisal of Ulva (Ulvophyceae,Chlorophyta) taxonomy

Journal of Applied Phycology - The green seaweed Ulva is important from ecological and economic perspectives, but the identification of species is often problematic. Here we assessed and discussed...     

Systematics of green algae resembling Ulva conglobata,with a description of Ulva adhaerens sp. nov. (Ulvales,Ulvophyceae)

A phylogenetic and morphological study of green algae resembling Ulva conglobata from Japan was undertaken, along with morphological observations of the original material of U. conglobata Kjellman. The samples resembling U. conglobata included five genetically distinct species: U. fasciata, U. pertusa, U. tanneri, Ulva sp. 1 and Ulva sp. 2. The discovery of marginal denticulations in some of the original material of U. conglobata, made it possible to distinguish those species without denticulations: U. pertusa, U. tanneri and Ulva sp. 2. The morphological characteristics of Ulva sp. 1 matched those of U. conglobata, but Ulva sp. 1 was not clearly identified as U. conglobata owing to the lack of DNA sequence data of the original material. Ulva sp. 2 had lobes adhering to each other by rhizoids. This morphological feature is stable in Ulva sp. 2 and unique among Ulva species. In conjunction with the molecular data, Ulva sp. 2 was described as a new species, U. adhaerens sp. nov. This species features rhizoidal extensions in regions other than the base and an elaborate arrangement of the extensions used for adhesion. It thereby expands our knowledge of the morphogenesis of the morphologically simple genus Ulva.     

Variability in the rbcL Introns of Caulerpalean Algae (Chlorophyta, Ulvophyceae)

rbcL gene have been reported to date. Four new cases from Caulerpales, Ulvophyceae are described here. In the genus Caulerpa, the presence of an intron was unstable even in the infraspecific taxa. Based on comprehensive comparisons of the inserted positions, lengths of introns and so on, the presence of at least three kinds of introns, which probably have independent origins, was suggested in Caulerpales. Received 12 May 2000/ Accepted in revised form 12 October 2000     

Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from <Emphasis Type="Italic">Enteromorpha prolifera</Emphasis> (Ulvophyceae,Chlorophyta)

Heat shock protein 70 (Hsp70) is one of the important members of heat shock protein (Hsp) families and plays essential roles in folding nascent protein, translocation, refolding denatured protein, protein degradation, adverse stress resistance, and so on. In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone full-length cytosolic heat shock protein 70 of Enteromorpha prolifera (designed as EPHsp70). Bioinformatics was used to analyze structural feature, homologous relationship, and phylogenetic position of EPHsp70. The full length of EPHsp70 cDNA was 2,265 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 217 bp, and an open-reading frame of 1,983 bp encoding a polypeptide of 660 amino acids with an estimated molecular weight of 71.39 kDa and an estimated isoelectric point of 5.03. EPHsp70 had five degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The C-terminus amino acid sequence of cytosolic EPHsp70 was EEVD, and the conservation of EPHsp70 of N-terminus was higher than that of C-terminus. The homology between EPHsp70 and the cytosolic Hsp70s of other algae and land plants was more than 70%.     

Antifouling potential of lubricious, micro-engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5 microns wide pillars or ridges separated by 5, 10, or 20 microns wide channels. Pattern depths were 5 or 1.5 microns. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5 microns wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5 microns, 10 microns and 20 microns. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.