Ludovic Delage

The Halogenated Metabolism of Brown Algae (Phaeophyta), Its Biological Importance and Its Environmental Significance

Brown algae represent a major component of littoral and sublittoral zones in temperate and subtropical ecosystems. An essential adaptive feature of this independent eukaryotic lineage is the ability to couple oxidative reactions resulting from exposure to sunlight and air with the halogenations of various substrates, thereby addressing various biotic and abiotic stresses i.e., defense against predators, tissue repair, holdfast adhesion, and protection against reactive species generated by oxidative processes. Whereas marine organisms mainly make use of bromine to increase the biological activity of secondary metabolites, some orders of brown algae such as Laminariales have also developed a striking capability to accumulate and to use iodine in physiological adaptations to stress. We review selected aspects of the halogenated metabolism of macrophytic brown algae in the light of the most recent results, which point toward novel functions for iodide accumulation in kelps and the importance of bromination in cell wall modifications and adhesion properties of brown algal propagules. The importance of halogen speciation processes ranges from microbiology to biogeochemistry, through enzymology, cellular biology and ecotoxicology.

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.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

A family of RRM-type RNA-binding proteins specific to plant mitochondria

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.

In vitro import of a nuclearly encoded tRNA into mitochondria of Solanum tuberosum.

ABSTRACT Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.

The T-domain of cytosolic tRNAVal, an essential determinant for mitochondrial import

Import of tRNAs into plant mitochondria appears to be highly specific. We recently showed that the anticodon and the D‐domain sequences are essential determinants for tRNAVal import into tobacco cell mitochondria. To determine the minimal set of elements required to direct import of a cytosol‐specific tRNA species, tobacco cells were transformed with an Arabidopsis thaliana intron‐containing tRNAMet‐e gene carrying the D‐domain and the anticodon of a valine tRNA. Although well expressed and processed into tobacco cells, this mutated tRNA was shown to remain in the cytosol. Furthermore, a mutant tRNAVal carrying the T‐domain of the tRNAMet‐e, although still efficiently recognized by the valyl‐tRNA synthetase, is not imported into mitochondria. Altogether these results suggest that mutations affecting the core of a tRNA molecule also alter its import ability into plant mitochondria.

Expression of a PKSIII Gene and soluble phlorotannin synthesis in response to abiotic and biotic stresses in the brown alga Fucus Vesiculosus: constitutive versus inductive protection

Station Biologique de Roscoff, Sorbonne Universités, Université Pierre et Marie Curie, CNRS, Roscoff 29688, France; Institut de Systématique, Evolution, Biodiversité, UMR 7205CNRS-EPHE-MNHN-UPMC, Muséum National d’Histoire Naturelle, Paris 75231, France; Station Biologique de Roscoff, CNRS, Sorbonne Universités, Université Pierre et Marie Curie, Roscoff 29688, France and Station Biologique de Roscoff, CNRS, Sorbonne Universités, Université Pierre et Marie Curie, Roscoff 29688, FranceEarly life-stage of the bladderwrack Fucus vesiculosus is highly influenced by the climate change factors temperature, CO2 and eutrophication. Intraspecific genetic diversity of Baltic Fucus vesiculosus populations is low, compared to e.g. Atlantic populations, which may limit their potential for adaptation. To nassess the role of intraspecific genetic diversity on the tolerance towards environmental change we manipulated their diversity: Plots with full-sibling Keynote and Oral Papers 106 Downloaded by [University of Kiel] at 02:13 22 September 2015 groups of Fucus germlings each originating from one parental pair represents the low diversity level, whereas plots with sibling groups from multiple parental pairs represent the high diversity level. Climate change was simulated according to the year 2100 in the near-natural scenario Kiel Benthocosms by maintaining the environmental fluctuations of the Baltic Sea and adding 5°C warming, 600 μatm pCO2 and doubling the nutrient concentrations. Germlings nresponded to warming with higher mortality and enhanced growth rates. High pCO2 concentrations nincreased growth due to a fertilisation effect. Nonphotochemical quenching was lower under warmed than ambient temperatures. A positive co-tolerance among sibling groups towards warming and acidification indicates the possible attenuation in presence of the multiple factors. Considerable differences among sibling group performance indicate a higher adaptive potential for genetically diverse populations. The high diversity levels also showed higher survival, indicating possible facilitation processes among genotypes. nMicrosatellite genotyping is in progress for revealing whether and how selection processes took place in high diversity levels. We conclude that impacts on early life-stage bladderwrack depend on the combination of stressors and season and that genetic variation nis crucial for local adaptation to climate change stress