Effects of ultraviolet radiation and temperature on the ultrastructure of zoospores of the brown macroalga Laminaria hyperborea

The interactive effects of an 8 h exposure to UV radiation and altered temperatures on the ultrastructure and germination of zoospores of the sublittoral brown alga Laminaria hyperborea (Gunn.) Foslie were investigated for the first time. Spores were exposed to four temperatures (2, 7, 12 and 17 degrees C) and three light regimes (PAR, PAR + UV-A, PAR + UV-A+UV-B). Freshly-released spores of L. hyperborea lack a cell wall and contain a nucleus with fine granular nucleoplasm and a nucleolus, one chloroplast, several mitochondria, dictyosomes and an endoplasmatic reticulum. Further, several kinds of so-called adhesive vesicles, lipid globuli and physodes containing UV-absorbing phlorotannins are embedded in the cytoplasm. No eye-spot is present. Physodes were found but they were rare and small. After an 8 h exposure to UV-B, the nucleoplasm had a mottled structure, chloroplasts contained plastoglobuli, the structure of the mitochondria changed from crista- to sacculus-type and germination was strongly inhibited at all temperatures. UV-A only had an impact on the ultrastructure at the highest temperature tested. The strongest effects were found at 17 degrees C, where germination was reduced to 35%, 32% and 9% after exposure to PAR, PAR+UV-A and PAR + UV-A + UV-B, respectively. This study indicates that UV-B radiation has strong damaging effects on the physiology and ultrastructure of zoospores of L. hyperborea. The results are important for developing scenarios for the effect of enhanced UV radiation and increasing temperatures caused by global climate changes.     

Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.     

Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata

Oligomeric degradation products of alginate elicited a respiratory and oxidative burst in the sporophytes of the kelp Laminaria digitata. The generation of activated oxygen species (AOS), O(2)(-), and H(2)O(2) was detected at the single cell level, using nitroblue tetrazolium precipitation and a redox-sensitive fluorescent probe, respectively. The oxidative burst involved diphenyleneiodonium-sensitive AOS-generating machinery and its amplitude depended on the type of tissue. After a first elicitation plants were desensitized for about 3 h. The activity of alginate oligosaccharides was dose dependent, saturating around 40 microM. It was also structure-dependent, with homopolymeric blocks of alpha-1,4-L-guluronic acid, i.e. the functional analogs of oligogalacturonic blocks in pectins, being the most active signals. The perception of oligoguluronate signals resulted in a strong efflux of potassium. Pharmacological dissection of the early events preceding the emission of AOS indicated that the transduction chain of oligoguluronate signals in L. digitata is likely to feature protein kinases, phospholipase A(2), as well as K(+), Ca(2+), and anion channels.     

The mitochondrial genome of the brown alga Laminaria digitata: a comparative analysis

We report here the complete sequence of the mitochondrial genome of the brown alga Laminaria digitata (Hudson) J.V. Lamouroux. L. digitata mtDNA is a circular molecule of 38,007?bp (64.9% A?+?T), encoding 63 genes and 3 ORFs and with only 6.7% of non-coding sequences. Based on gene content and order, its overall organization is very similar to that of the mitochondrial genome of Pylaiella littoralis, another brown alga belonging to a different sublineage of the Phaeophyceae. In particular, the two genomes share unusual features, which hence could be unique to brown algae among the heterokont lineage, namely the presence of a rn5 gene, a short nad11 gene, a cox2 gene with a large in-frame insertion and α-proteobacterial-like promoter sequences. On the other hand, L. digitata lacks the sequences which are thought to have been transmitted horizontally to the P. littoralis genome, that is, the group-II introns in the rnl and cox1 genes, and it features only traces of an ancestral T7-like RNA polymerase. Distance phylogenetic trees inferred from concatenated mitochondrial genes confirm that speciation of brown algae occurred recently compared to other heterokonts.     

Impact of ultraviolet radiation on physiology and development of zoospores of the brown alga Alaria esculenta from Spitsbergen

Zoospores of the brown alga Alaria esculenta from Spitsbergen were exposed in the laboratory to photosynthetically active radiation [(P), 400–700 nm], P + UVA radiation (PA, 320–700 nm) and PA + UVB radiation (PAB, 280–700 nm). Germination rates were determined, and the germination process was documented by light microscopy. In parallel, the presence of UV-absorbing phlorotannins was studied. Photoinhibition and recovery of photosynthesis were monitored as well as DNA damage and repair. After 8 h of exposure to PAB, germination was inhibited. A 16-h exposure to PA and PAB resulted in a dramatic reduction of germination rates. Phlorotannin-containing physodes were observed in the peripheral cytoplasm and physode-like bodies were found outside the spore, still attached to the zoospore as well as freely floating in the medium; this suggested exocytosis. The absorbance of spore suspensions below 300 nm was higher after 20 h exposure to P, PA and PAB compared with the dark treatment. About 50% of the radiation was absorbed by the zoospores, and the rest was absorbed by the medium, especially after PA and PAB exposure. In this way, harmful UV radiation is absorbed before reaching the cells and this is probably the major reason for the relatively low DNA damage after ≤8 h exposure to PAB. Under these conditions, DNA damage was efficiently repaired and there was a notable recovery of photosynthesis. However, after 16 h exposure to PA and PAB, the protective and repair mechanisms are surcharged and the zoospores degenerate. The results cast light on the potential impact of enhanced UVB radiation on a dominant component of the seaweed community on Spitsbergen because of stratospheric ozone depletion. The acclimation potential of zoospores of this species to UV radiation as demonstrated here is regarded as conferring a competitive advantage in recruitment over similar species in the upper sublittoral zone.     

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.     

The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities

Different haloperoxidases, one specific for the oxidation of iodide and another that can oxidize both iodide and bromide, were separated from the sporophytes of the brown alga Laminaria digitata and purified to electrophoretic homogeneity. The iodoperoxidase activity was approximately seven times more efficient than the bromoperoxidase fraction in the oxidation of iodide. The two enzymes were markedly different in their molecular masses, trypsin digestion profiles, and immunological characteristics. Also, in contrast to the iodoperoxidase, bromoperoxidases were present in the form of multimeric aggregates of near-identical proteins. Two full-length haloperoxidase cDNAs were isolated from L. digitata, using haloperoxidase partial cDNAs that had been identified previously in an Expressed Sequence Tag analysis of the life cycle of this species (1). Sequence comparisons, mass spectrometry, and immunological analyses of the purified bromoperoxidase, as well as the activity of the protein expressed in Escherichia coli, all indicate that these almost identical cDNAs encode bromoperoxidases. Haloperoxidases form a large multigenic family in L. digitata, and the potential functions of haloperoxidases in this kelp are discussed.     

Kinetics modeling of alginate alkaline extraction from Laminaria digitata

Alginates being depolymerized during their alkaline extraction, reducing extraction time could help producing higher rheological quality alginates. The purpose of the present work is to study fresh Laminaria digitata destructuration during alkaline extraction and its link to extraction kinetics. Both alginate extraction yield and mean diameter of algae particles were followed for different values of agitation level and initial size of algae pieces. Results highlighted the existence of a link between extraction yield and algal destructuration. Those elements and the specificity of L.digitata structure have been taken into account to propose a kinetics model based on a fluid-particle reaction with decreasing size particles. The model parameters have been adjusted thanks to acquisition data and its predictive capacity was assessed by validation data. Provided predictions appeared to be relevant and the model structure suitability was confirmed, as extraction yield kinetics specific shape was quite reliably described.     

Chromosome Number of Laminaria digitata Lamour

An investigation of the chromosome number of Laminaria digitataLamour has revealed the diploid complement to be 16. Endomitoticchromosome multiplication has been observed. A similar investigationby Magne has shown the diploid number of chromosomes to be 26in Laminaria flexicaulis Le Jolis and raises the taxonomicalquestion whether these two names can be regarded as synonymsas at present used or covering distant species.     

Waterborne signaling primes the expression of elicitor-induced genes and buffers the oxidative responses in the brown alga Laminaria digitata

As marine sessile organisms, seaweeds must respond efficiently to biotic and abiotic challenges in their natural environment to reduce the fitness consequences of wounds and oxidative stress. This study explores the early steps of the defense responses of a large marine brown alga (the tangle kelp Laminaria digitata) and investigates its ability to transmit a warning message to neighboring conspecifics. We compared the early responses to elicitation with oligoguluronates in laboratory-grown and harvested wild individuals of L. digitata. We followed the release of H₂O₂ and the concomitant production of volatile organic compounds. We also monitored the kinetics of expression of defense-related genes following the oxidative burst. Laboratory-grown algae were transplanted in kelp habitats to further evaluate their responses to elicitation after a transient immersion in natural seawater. In addition, a novel conditioning procedure was established to mimic field conditions in the laboratory. Our experiments showed that L. digitata integrates waterborne cues present in the kelp bed and/or released from elicited neighboring plants. Indeed, the exposure to elicited conspecifics changes the patterns of oxidative burst and volatile emissions and potentiates this kelp for faster induction of genes specifically regulated in response to oligoguluronates. Thus, waterborne signals shape the elicitor-induced responses of kelps through a yet unknown mechanism reminiscent of priming in land plants.     

Microchemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation

Brown algal kelp species are the most efficient iodine accumulators among all living systems, with an average content of 1.0% of dry weight in Laminaria digitata. The iodine distributions in stipe and blade sections from L. digitata were investigated at tissue and subcellular levels. The quantitative tissue mapping of iodine and other trace elements (Cl, K, Ca, Fe, Zn, As and Br) was provided by the proton microprobe with spatial resolutions down to 2 μm. Chemical imaging at a subcellular resolution (below 100 nm) was performed using the secondary ion mass spectrometry microprobe. Sets of samples were prepared by both chemical fixation and cryofixation procedures. The latter prevented the diffusion and the leaching of labile inorganic iodine species, which were estimated at around 95% of the total content by neutron activation analysis. The distribution of iodine clearly shows a huge, decreasing gradient from the meristoderm to the medulla. The contents of iodine reach very high levels in the more external cell layers, up to 191 ± 5 mg g⁻¹ of dry weight in stipe sections. The peripheral tissue is consequently the main storage compartment of iodine. At the subcellular level, iodine is mainly stored in the apoplasm and not in an intracellular compartment as previously proposed. This unexpected distribution may provide an abundant and accessible source of labile iodine species which can be easily remobilized for potential chemical defense and antioxidative activities. According to these imaging data, we proposed new hypotheses for the mechanism of iodine storage in L. digitata tissues. In memory of Dr. Charles Mioskowski, “Miko,” who died on 2 June 2007.     

EST-SSR markers derived from Laminaria digitata and its transferable application in Saccharina japonica

The public availability of numerous expressed sequence tag (EST) enables EST-based SSR (simple sequence repeat) markers to be widely used for genetics and breeding studies. In the present study, EST-SSR markers were developed from ESTs of Laminaria digitata and were transferred to the non-congeneric species Saccharina japonica. Among the 2,668 non-redundant ESTs, 83 (3.1%) ESTs containing SSR were identified totally, with an average of one SSR per 13.6 kb. Analysis of SSR motifs revealed that the trinucleotide and tetranucleotide were major motifs, accounted for 44.58% and 16.87%, respectively. Based on the 83 ESTs containing SSR, we designed 45 pairs of primers in the flanking regions of the SSR, of which 13 pairs showed polymorphism in a wild S. japonica population, and the mean alleles per locus was 3.6 (ranging from 2 to 6). The observed (Ho) and expected (He) heterozygosities of these EST-SSRs were 0.234–0.632 and 0.260–0.635, respectively. All loci were in Hardy–Weinberg equilibrium in the wild population and no linkage disequilibrium was detected among loci. The obtained EST-SSR markers can facilitate and promote related research such as ecological investigation, genetic diversity assessment and breeding practice of S. japonica as well.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.