Isolated, distal blade discs of the brown alga Laminaria digitata form sorus, but not discs, near to the meristematic transition zone

Blade discs of vegetative thalli of Laminaria digitata (Huds.) Lamour. from Helgoland (North Sea) cut at 5–15 cm distance from the blade/stipe transition, formed sorus in the laboratory after 7–12weeks, 5 months earlier than whole fronds in the field. Sorus formation occurred in a broad range of daylength regimes or temperatures, at 8–16 h light pe rday and 6–12 ^°C. No sorus was developed during three months by meristematic blade discs cut from the lowermost 3 cm portion of the blade, nor from whole thalli cultured in parallel to isolated blade discs. These findings point to the possible existence of sporulation inhibitors produced by the laminarian meristem. This revised version was published online in August 2006 with corrections to the Cover Date.     

A comparative analysis of wild and laboratory grown Laminaria abyssalis (Phaeophyta) using modulated fluorescence

Laminaria abyssalis fronds were either collected at the Brazilian costal area - 40 meters below sea level - or grown in the laboratory. The photochemical yield as defined by the Fv/Fm and the Fo - the dark fluorescence level when all PSII centers are open - varied with the distance from the stipe to the tip of the blade in wild grown fronds while it stayed constant in the laboratory grown plants. The chlorophyll a/c ratio levels decreased in the wild fronds from 12 (near the stipe) to 6 near the top. The chlorophyll c content increased from 0.8 to near 1.7 mg cm^–2 in the wild fronds. The laboratory fronds did not show variations in their chlorophyll contents. The wild fronds pattern changed after 2 months kept in the laboratory, producing similar results to those grown in the laboratory. The results indicate that the levels of the antenna complex in the wild fronds increase from the stipe to the top of the blade, in a fashion similar of the sun/shade leaves. Also, results show, that this alga is able to adapt itself to new light conditions, possibly increasing its level of antenna complex and photosynthetic units.Abbreviations PSII Photosystem II - Fo Chlorophyll fluorescence when all PSII are opened - Fm Chlorophyll Fluorescence when all PSII are closed - Fv Variable Fluorescence (Fm-Fo) - Fv/Fm Quantum Yield for Photochemistry     

Development of 18 polymorphic microsatellite DNA markers of Laminaria japonica (Phaeophyceae)

Eighteen polymorphic microsatellite DNA markers were developed for Laminaria japonica, a brown alga cultured intensively in China. These markers are independent from each other and are moderately variable in L. japonica. The number of alleles and gene diversity detected in 53 gametophyte clones representing the varieties of L. japonica cultured once or currently in China range from two to nine and from 0.355 to 0.768, respectively. These markers will certainly facilitate the management and exploitation of the germplasm resource of L. japonica conserved indoor as gametophyte clones and the determination of the genetic diversity of L. japonica naturally distributed.     

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase

Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x10⁶ cells g^–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl₂, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW Flesh weight - AAP Abalone acetone powder - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hyrdoxymethyl)aminomethane - PESI Provasoli's enriched seawater with iodine     

Effect of temperature and photon fluence rate on gametophytes and young sporophytes of Laminaria ochroleuca Pylaie

We analysed the effects of temperature and photon fluence rate on meiospore germination, growth and fertility of gametophytes, and growth of young sporophytes of Laminaria ochroleuca. Maximum percentages of germination (91–98%) were obtained at 15°C and 18°C, independent of photon fluence rate. Optimal development of female gametophyte and maximum fecundity and reproductive success of gametophytes occurred at 15°C and 18°C and at 20 and 40 μmol m^–2 s^–1. Maximum relative growth rate of young sporophytes after 2 weeks of culture was achieved under the same conditions. L. ochroleuca gametophytes cannot reproduce and growth of its sporophytes is not competitive at temperatures close to 10°C. Received in revised form: 31 August 2001 Electronic Publication     

Nucleotide sequence diversity of the 5S rDNA spacer in the simple blade kelp genera Laminaria, Cymathaere and Kjellmaniella (Laminariales, Phaeophyceae) from northern Japan

Tandem repeats of the 5S ribosomal RNA gene (rDNA) were confirmed for almost all laminarian, cymathaerean and kjellmaniellan species distributed in northern Japan. The nucleotide sequence of the spacer region between tandemly repeated 5S rDNA was investigated for 79 samples from 31 sites. Phylogenetic analysis of the 29 different sequences detected revealed two lineages: (1) Laminaria coriacea group, including Laminaria coriacea Miyabe, Laminaria cichorioides Miyabe, Laminaria sachalinensis (Miyabe) Miyabe, Laminaria yendoana Miyabe, Cymathaere japonica Miyabe et Nagai, Kjellmaniella gyrata (Kjellman) Miyabe and Kjellmaniella crassifolia Miyabe; (2) Laminaria japonica group including Laminaria japonica Areschoug, Laminaria religiosa Miyabe, Laminaria ochotensis Miyabe, Laminaria diabolica Miyabe, Laminaria longipedalis Okamura, Laminaria angustata Kjellman and Laminaria longissima Miyabe. In addition, the latter group was divided into two: subgroup (2a) including L. angustata and L. longissima and subgroup (2b) including L. japonica, L. religiosa, L. ochotensis, L. diabolica and L. longipedalis. Members of the three groups differ from each other in the appearance of ornaments (bullation, gyration and folds) on the surface of the blade. These are absent in group (2a), only present in the early stages of the lifespan of group (2b), and present for the duration of the lifespan in group (1). Genetic distances among samples were extremely small within group (2a). Together with previous crossing studies and data on ocean currents and distribution, these findings suggest that gene flow occurs within group (2b).     

Kinetics of non-photosynthetic callus production from the brown alga Laminaria setchellii (Laminariales)

White, filamentous, callus-like tissue was produced from the temperate marine brown alga Laminaria setchellii Silva under non-photosynthetic conditions. Explant disks from the medullary core of the stipe section were plated on solid medium (1.5 wt% agar in seawater base, pH 8) at 8 ± 2°C in the dark, and the formation of callus-like tissue was followed as a function of time up to 8 weeks. Although 30% of the total explants plated formed filamentous, callus-like growths, only 3 to 6% of the total explants produced relatively large amounts of filamentous and rounded callus-like cells. The fresh weight yield of this callus-like tissue was 3.74 ± 0.93 mg per 10 mm explant disk (0.053 g callus per g explant). Attempts at establishing a growing, cell suspension of the dispersed callus tissue in PES liquid medium at 24 μmol photon m^?2 s^?1 were not successful.     

Taxonomy of the southwestern Atlantic endemic kelp: Laminaria abyssalis and Laminaria brasiliensis (Phaeophyceae,Laminariales) are not different species

Two endemic species of Laminaria, Laminaria abyssalis Joly & Oliveira Filho and L. brasiliensis Joly & Oliveira Filho, from the tropical southwestern Atlantic coast have been described. The aim of this work was to determine the conspecificity of these species based on morphological and molecular analyses (ribulose 1,5‐bisphosphate carboxylase/oxgenase, large subunit (rbcL), internal transcribed spacer (ITS) and cytochrome c oxidase subunit I (coxI)). We found an overlap between the morphological characters that are considered taxonomically important for distinguishing these two species; these characters included a differing pattern of blade splitting. In the three molecular analyses, the Brazilian Laminaria specimens were grouped into one clade with maximum support. These data support the hypothesis that the individuals analyzed represent only one species, L. abyssalis. The molecular analysis also showed L. abyssalis to be sister group to L. digitata.     

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.     

Thermal acclimation and photoacclimation of photosynthesis in the brown alga Laminaria saccharina

Thermal acclimation and photoacclimation of photosynthesis were compared in Laminaria saccharina sporophytes grown at temperatures of 5 and 17 °C and irradiances of 15 and 150μmol photons m^?2 s^?1. When measured at a standard temperature (17°C), rates of light-saturated photosynthesis (P_max) were higher in 5 °C-grown algae (c. 3.0 μmol O₂ m^?2 s^?1) than in 17 °C-grown algae (c. 0.9 μmol O₂ m_-2 s_-1). Concentrations of Rubisco were also 3-fold higher (per unit protein) in 5 °C-grown algae than in algae grown at 17 °C. Light-limited photosynthesis responded similarly to high temperature and low light Photon yields (α) were higher in algae grown at high temperature (regardless of light), and at 5 °C in low light, than in algae grown at 5 °C in high light Differences in a were correlated with light absorption; both groups of 17 °C algae and 5 °C low-light algae absorbed c. 75% of incident light, whereas 5 °C high-light algae absorbed c. 55%. Increased absorption was correlated with increases in pigment content PSII reaction centre densities and the fucoxanthin-Chl ale protein complex (FCP). Changes in a were also attributed, in part, to changes in the maximum photon yield of photosynthesis (0_max). PSI reaction centre densities were unaffected by growth temperature, but the areal concentration of PSI in low-light-grown algae was twice that of high-light-grown algae (c. 160.0 versus 80.0 nmol m^?2). We suggest that complex metabolic regulation allows L, saccharina to optimize photosynthesis over the wide range of temperatures and light levels encountered in nature.     

Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica

A low molecular weight sulfated polysaccharide (L-A) was prepared bymild acid hydrolysis of crude fucoidan (F-A) from Laminaria japonica. In comparison with F-A, L-A had a lower proportion of fucose residues,but a similar proportion of sulfate. The galactose content of both fractionswas relatively high, especially for L-A (57%). The antioxidant propertiesof the two polysaccharides were studied using two low-density lipoproteinoxidation systems. L-A had a stronger effect against low-density lipoproteinoxidation in both systems. F-A inhibited the AAPH-induced low-densitylipoprotein oxidation, but had little effect on the Cu⁺⁺-inducedsystem due to its large molecular mass. The active sulfated fraction L-A,containing galactose, mannose and fucose (about 9: 2: 2) is reported herefor the first time.     

Inducible effects of abscisic acid on sporophyte discs from Laminaria japonica Areschoug (Laminariales, Phaeophyceae)

The effects of exogenous abscisic acid (ABA;10^–7–10^–5 M), a knownplantgrowth regulator, on reproduction and growth were investigated by culturingdiscs from sporophytes of Laminaria japonica Areschoug.ABAplays a role in triggering sorus formation, and it was found that sorusformation of discs was fastest in 10^–5 M ABA. Theapplication of 10^–5 M ABA to culturing discs alsosuppressed the expansion of surface area. ABA contents in sorus and vegetativeparts of the sporophyte were determined by bioassay. The mean ABA content insorus parts obtained from sporophytes was 0.222 ± 0.053g equivalent-ABA g wet weight^–1, which wasabout five times higher than the content found in vegetative parts (0.048± 0.009 g equivalent-ABA g wetweight^–1). Taken together these results suggest that sorusdevelopment requires an elevated level of ABA and is associated with decreasingvegetative growth and that the ABA level of the sporophyte may play a crucialrole in reproduction.     

Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases

Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg²⁺ and Sr²⁺ in addition to Ca²⁺ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 μm, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.     

Isolation and structural elucidation of a growth stimulant for arbuscular mycorrhizal fungus from Laminaria japonica Areschoug

Arbuscular Mycorrhizal (AM) fungi enhance terrestrial plant growth by forming a symbiotic relationship with the roots of its host plant. A growth stimulant for AM fungi was isolated from a brown alga Laminaria japonica Areschoug. The active substance for in vitro AM hyphal growth was isolated from 75% methanol extracts of L. japonica using a succession of chromatographic procedures, including flash chromatography equipped with an octa decyl silane (ODS) column, gel filtration chromatography and HPLC using an ODS column. Spores of Gigaspora margarita Becker & Hall, an AM fungus, were exposed to the compound in vitro, and hyphal growth of G. margarita was measured after two weeks of incubation. At 40 mg L⁻¹, the compound significantly stimulated the in vitro hyphal growth of G. margarita, compared to the control. This compound was elucidated as 5′-deoxy-5′-methylamino-adenosine by EIMS and NMR spectrum.     

Influence of Laminaria japonica added to the diet on remote effects of radiation injuries

White rats were irradiated by both ¹³¹iodine, which was incorporated into the thyroid gland, and ¹³⁷cesium; these were dosed at about 10 and 6 Gy, respectively. A portion of the experimental animals was fed a diet enriched with powdered Laminaria japonica of known composition. In animals provided with this powder, the frequency of neoplasms was lessened, and the latent period for the major types of tumours was increased, compared to rats not given the powder. (*author for correspondence)     

Incorporation of 14C-radioactivity into lipid classes at different maturity of Laminaria japonica Aresch. cultured under natural and partially controlled conditions

A study to assess which environmental or developmental factors predominate in the biosynthesis of lipids of Laminaria japonica Aresch. blades was undertaken by means of ¹⁴C-labelling technique. In experiment 1, kelp blades at different growth stages were collected in different cultural seasons. In experiment 2, kelp blades of different sizes and maturity cultured simultaneously for two months in the same sea area were collected at the same time.The following results were obtained. In experiment 1, the ¹⁴C-incorporation into whole lipids was lowest in juvenile blades collected at the end of autumn and highest in blades of middle size collected in winter. However, the highest counts were incorporated in PC among complex lipid classes from all size classes of blades in both experiments 1 and 2. In experiment 2, ¹⁴C-incorporation patterns of individual lipid classes were characteristically different depending on the sizes of blades even under the same cultural condition. Thus, the biosynthesis of lipids in this kelp seems to be affected essentially by developmental factors.Abbreviation Comp. lip. complex lipid - FA non-esterified fatty acids - Fucost fucosterol - DG diacylglycerol - DGDG digalactosyldiacylglycerol - MG monoacylglycerol - MGDG monogalactosyldiacylglycerol - NL neutral lipids - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidyl inositol - PS phosphatidylserine - SQDG sulphoquinovosyldiacyl glycerol - TG triacylglycerol     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

A simple method for DNA extraction from sporophyte in the brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis>

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol (24:1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 μg g⁻¹ (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A₂₆₀/A₂₈₀, which was about 1.7–1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K _m ^I– =2.5 mM; k _cat ^I– =462 s^–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K _m ^I– =18.1 mM; k _cat ^I– =38 s^–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804.     

Evaluation of the potential role of the macroalga Laminaria japonica for alleviating coastal eutrophication

The rapid development of human activities has caused serious eutrophication of coastal waters in China in the recent decades. The study of the biofiltration capacity of Laminaria japonica under laboratory conditions showed a significant nutrient uptake. After 36 h of incubation, around 42%, 46%, 44% of N and 45%, 42%, 35% of P were removed from three gradients of medium concentrations, respectively. In the conditions of different ratios of N/P and NO₃–N/NH₄–N, the optimum N/P ratio for nutrient uptake was 7.4 and L. japonica prefered NO₃–N rather than NH₄–N as nitrogen source. Temperature and irradiance affected uptake rates significantly. The maximal N uptake rate appeared at 10 °C and 18 μmol photons m⁻² s⁻¹ and the maximal P uptake rate was found at 15 °C and 144 μmol photons m⁻² s⁻¹. Moreover, further studies were needed to investigate the bioremediation potential of L. japonica in the open sea.