PHYLOGENY AND SYSTEMATICS OF THE MARINE ALGAL FAMILY GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) BASED ON SMALL SUBUNIT rDNA AND ITS SEQUENCES OF ATLANTIC AND PACIFIC SPECIES1

We sequenced the small subunit rDNA and internal transcribed spacer region of Gracilariaceae from the tropical Atlantic and Pacific, with emphasis on flattened or compressed species. Sequence comparisons confirmed three main lineages of Gracilariaceae: Curdiea/Melanthalia, Gracilariopsis/Gracilariophila, and Gracilaria. The Curdiea/Melanthalia diverged early in the family. Gracilariopsis was paraphyletic, because at least one Gracilariophila species evolved from it. The Atlantic Gracilariopsis were monophyletic and separated from the Pacific lineages. The Gracilaria included all species referable to its own species and to Hydropuntia, which was paraphyletic, formed by distantly related lineages. The new combination Gracilaria pauciramosa (N. Rodríguez Ríos) Bellorin, M. C. Oliveira et E. C. Oliveira is proposed for Polycavernosa pauciramosa N. Rodríguez Ríos. Recognition of subgenera within Gracilaria, based on spermatangial arrangement, was not supported. Instead, infrageneric groups were delineated by geographic origins and combinations of reproductive characters. Most Pacific species with either “textorii” or “verrucosa” type spermatangia were deeply separated from Atlantic species. Within the Atlantic Gracilaria, a lineage encompassing mostly tropical cylindrical species with “henriquesiana” type spermatangia and distinctive cystocarp anatomy was recognized. A lineage was also retrieved for cold water stringy species with verrucosa type spermatangia. Several species from the western Atlantic are closely related to Gracilaria tikvahiae McLachlan with nearly identical morphology. On the other hand, most flattened species from the tropical Atlantic were closely related despite their diverse morphologies. The interpretation of our data in addition to the literature indicates that more populations from the Indo‐Pacific must be studied before a general picture of Gracilariaceae evolution can be framed.     

Glutamine Synthetase (GS): A Key Enzyme for Nitrogen Assimilation in The Macroalga Gracilariopsis lemaneiformis (Rhodophyta)

This study aimed to address the importance of glutamine synthetase II (GSII) during nitrogen assimilation in macroalga Gracilariopsis lemaneiformis. The cDNA full‐length sequence of the three glGSII genes was revealed to have the 5′ m⁷G cap, 5′‐untranslated region, open reading frame (ORF), 3′‐untranslated region, and a 3′ poly (A) tail. The three glGSIIs were classified into plastid glGS2 and cytosolic glGS1‐1 and glGS1‐2, having conserved GSII domains but different cDNA sequences. The complicated 5′ end flanking region indicates complex function of glGS genes. glGS1 genes were significantly up‐regulated under the different NH₄⁺: NO₃^? ratio (i.e., 40:10, 25:25, 10:40, and 0:50) except glGS2 which dramatically up‐regulated under the low NH₄⁺: NO₃^? ratio (i.e., 10:40 and 0:50) during different cultivation times. These different expression patterns perhaps are due to the different biological roles of GS1 and GS2 in the gene family. Furthermore, hypothetical working model of nitrogen assimilation pathway exhibiting the role of glGS1 and glGS2 is proposed. Finally, glGS2 was expressed in Escherichia coli BL21 (DE3), and the optimal conditions for culture (15°C, overnight), purification (500 mM imidazole washing), and activity (pH 7.4, 37°C) were established. This study lays a very important foundation for exploring the role of GS in nitrogen assimilation in algae and plants.     

Yield, chemical composition and gel strength of agarocolloids of Gracilaria gracilis, Gracilariopsis longissima and the newly reported Gracilaria cf. vermiculophylla from Roscoff (Brittany, France)

Agarocolloids were extracted from field samples of Gracilaria gracilis, Gracilariopsis longissima and the newly reported Gracilaria cf. vermiculophylla harvested at different periods of the year near Roscoff (France). Native and alkali modified extracts were characterized by GLC, HPLC and FT-IR spectroscopy. The main components of agarocolloids isolated by freeze-thawing method, were 3,6-anhydrogalactose and galactose. In addition, minor components (6-O-methyl-galactose, 4-O-methyl-galactose and sulfate groups ranging from 4.4 up to 6.6% [w/w]) were detected. The highest rate of 6-O-methylgalactose was observed in agarocolloids from vermiculophylla (14 mole%). Sulfates were mainly branched on C4 of the D-galactose in gracilis and Gs. longissima agarocolloids. G. vermiculophylla agaroids isolated by EtOH and NaCl precipitations from the syneresis water were characterized by a high sulfation on C6 of galactose and a low sulfation on C2 of 3,6-anhydrogalactose. Native agarocolloid gel strengths from Gracilaria species were clearly higher than those of Gracilariopsis. Alkali treatments reduced the sulfate levels but increased slightly the gel strengths. An approximation of the polymer sizes carried out with colorimetric assays indicated that the polymer sizes were higher in G. gracilis than observed in Gs. longissima. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro anti-influenza virus activities of sulfated polysaccharide fractions from <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis>

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Structure of reproductive apparatus of Gracilaria/ Gracilariopsis lemaneiformis (Gracilariaceae, Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released. Supported by the National Natural Sciences Foundation of China (Grant No. 40606034) and the National High-Tech Research and Development Program of China (Grant No. 2006AA10A413)     

Structure of reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial devel-opment revealed the similar cytodynamic pattern followed by all the Gracilariaceae members devel-oped from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.     

Effect of visible light on superoxide dismutase (SOD) activity in the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)

Superoxide dismutase (SOD) was studied in the agarophyte Gracilariopsis tenuifrons. Similar SOD activity (130 ± 9U mg^-1) was observed in material from different regions of SouthAmerica, from different phases of the life cycle (gametophytes andtetrasporophytes), and from apical and basal sections of the thallus.In alga grown under a light-dark cycle, SOD activity in samples takenat different times exhibited a diurnal rhythm. The activity measured duringthe day phase was twice as much as during the night phase. This rhythm didnot persist under constant light, indicating light regulation of SOD activity.SOD activity was tested in algae submitted to different light intensities anddifferent wavelengths. It increased with the light intensity. The blue lightwavelength exerted a greater induction of SOD activity than other specificwavelengths.