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Brown algae are one of the few eukaryotic lineages that have evolved complex multicellularity, together with Opisthokonts (animals, fungi) and Plantae (land plants, green and red algae). In these three lineages, biotic stresses induce similar local defense reactions. Animals and land plants also feature a systemic immune response, protecting the whole organism after an attack on one of its parts. However, the occurrence of systemic defenses has never been investigated in brown algae. We elicited selected parts of the kelp Laminaria digitata and monitored distant, nonchallenged areas of the same individual for subsequent defense reactions. A systemic reaction was detected following elicitation on a distant area, including an oxidative response, an increase in haloperoxidase activities and a stronger resistance against herbivory. Based on experiments with pharmacological inhibitors, the liberation of free fatty acids is proposed to play a key role in systemic signaling, reminiscent of what is known in land plants. This study is the first report, outside the phyla of Opisthokonts and Plantae, of an intraorganism communication leading to defense reactions. These findings indicate that systemic immunity emerged independently at least three times, as a consequence of convergent evolution in multicellular eukaryotic lineages., (© 2014 European Union. New Phytologist © 2014 New Phytologist Trust.)

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant - the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide.

Arachidonic acid, linolenic acid and methyl jasmonate (MeJA) were found to be strong triggers of an oxidative burst in the kelp Laminaria digitata. These findings constitute the first report of an oxidative burst in an algal system induced by free fatty acids. The source of reactive oxygen species can be at least partially inhibited by diphenylene iodonium (DPI). Treatment with arachidonic acid increases the levels of a number of free fatty acids [including myristic (C14:0), linoleic (C18:2), linolenic (C18:3) and eicosapentaeneoic (C20:5) acids] and hydroxylated derivatives [such as 15-hydroxyeicosatetraenoic acid (15-HETE), 13-hydroxyoctadecatrienoic acid (13-HOTE) and 15-hydroxyeicosapentaenoic acid (15-HEPE)]. Similar to a previous report of the function of an alginate oligosaccharide-triggered oxidative burst in the establishment of resistance in L. digitata against infection by its brown algal endophyte Laminariocolax tomentosoides, C20:4- and MeJA-induced oxidative bursts seem to be involved in establishing the same protection in L. digitata. Altogether, this study supports the notion that lipid oxidation signaling plays a key role in defense induction in marine brown algae.

Alginate is an industrially important polysaccharide obtained commercially by harvesting brown algae. The final step in alginate biosynthesis, the epimerization of beta-1,4-d-mannuronic acid to alpha-1,4-l-guluronic acid, a structural change that controls the physicochemical properties of the alginate, is catalyzed by the enzyme mannuronan C-5-epimerase. Six different cDNAs with homology to bacterial mannuronan C-5-epimerases were isolated from the brown alga Laminaria digitata (Phaeophyceae). Hydrophobic cluster analysis indicated that the proteins encoded by the L. digitata sequences have important structural similarities to the bacterial mannuronan C-5-epimerases, including conservation of the catalytic site. The expression of the C-5-epimerase genes was examined by northern-blot analysis and reverse transcriptase-polymerase chain reaction in L. digitata throughout a year. Expression was also monitored in protoplast cultures by northern and western blot, reverse transcriptase-polymerase chain reaction, and activity measurements. From both the structural comparisons and the expression pattern, it appears that the cDNAs isolated from L. digitata encode functional mannuronan C-5-epimerases. The phylogenetic relationships of the bacterial and brown algal enzymes and the inferences on the origin of alginate biosynthetic machinery are discussed.

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.

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.

Laminariales (Phaeophyceae, Heterokonta) are characterised by a heteromorphic digenetic life cycle with a filamentous, microscopic gametophyte and a highly evolved, macroscopic sporophyte. With the ultimate goal of comparing gene expression in each life cycle stage, complementary DNA libraries were constructed from sporophytes and gametophytes of Laminaria digitata. A set of ca. 500 expressed sequence tags (EST) was generated from each life history phase, by single-run partial sequencing of randomly picked cDNA clones. Comparison of the EST deduced amino acid sequences with database protein sequences assigned a putative identity for 39% of the 412 gametophyte clones and 48% of the 493 sporophyte clones sequenced thus far. These data represent more than 152 different proteins now probably identified in L. digitata. Several of those newly identified proteins are of interest to our understanding of the molecular physiology of kelps, for example their carbon-concentrating mechanisms, cell wall biosynthesis and halogen metabolism. EST analysis also confirmed that genes with long 3'-UTRs are widespread in Laminariales and the study of 5'-UTRs allowed the identification of a Kozak consensus sequence, c(A/C)A(A/C)CAUGGc(G/T). Several potential developmentally regulated differences in gene expression are discussed.

Random Amplified Polymorphic DNA (RAPD) markers were developed for genotyping in Laminaria digitata. Reproducible RAPD banding patterns were reliably obtained, using a quick micro­scale DNA extraction procedure. Intraspecific polymorphism was observed, allowing for unambiguous genotyping within sets of 3 4 sporophytes. A controlled crossing indicated that in this kelp RAPD markers are transmitted to the progeny in a Mendelian way. This approach was then applied to parentage analysis within a set of 30 anonymous sporophytes from a random cross experiment involving 4 parents. The parents of 26 offsprings were unambiguously identified, indicating that cross­fertilisation largely, if not totally, prevailed over self­fertilisation. [ABSTRACT FROM AUTHOR]