Specificity of a ß-porphyranase produced by the carrageenophyte red alga Chondrus crispus and implications of this unexpected activity on red algal biology.

The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial ß-carrageenase and ß-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected ß-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a KM of 4.0 ± 0.8 µM, a kcat of 79.9 ± 6.9 s-1, and a kcat/KM of 20.1 ± 1.7 µM-1 s-1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This ß-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization.

To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.

Chondrus crispus is a marine red alga with sulfated galactans, called carrageenans, in its extracellular matrix. Chondrus has a complex haplodiplontic life cycle, alternating between male and female gametophytes (n) and tetrasporophytes (2n). The Chondrus life cycle stages are isomorphic; however, a major phenotypic difference is that carrageenan composition varies significantly between the tetrasporophytes (mainly lambda-carrageenan) and the gametophytes (mainly kappa/iota-carrageenans). The disparity in carrageenan structures, which confer different chemical properties, strongly suggests differential regulation of carrageenan-active genes between the phases of the Chondrus life cycles. We used a combination of taxonomy, biochemistry and molecular biology to characterize the tetrasporophytes and male and female gametophytes from Chondrus individuals isolated from the rocky seashore off the northern coast of France. Transcriptomic analyses reveal differential gene expression of genes encoding several galactose-sulfurylases, carbohydrate-sulfotransferases, glycosyltransferases, and one family 16 glycoside hydrolase. Differential expression of carrageenan-related genes was found primarily between gametophytes and tetrasporophytes, but also between the male and female gametophytes. The differential expression of these multigenic genes provides a rare glimpse into cell wall biosynthesis in algae. Furthermore, it strongly supports that carrageenan metabolism holds an important role in the physiological differentiation between the isomorphic life cycle stages of Chondrus.

Characterization of Nme5-Like Gene/Protein from the Red Alga Chondrus Crispus.

The Nme gene/protein family of nucleoside diphosphate kinases (NDPK) was originally named after its member Nm23-H1/Nme1, the first identified metastasis suppressor. Human Nme proteins are divided in two groups. They all possess nucleoside diphosphate kinase domain (NDK). Group I (Nme1-Nme4) display a single type NDK domain, whereas Group II (Nme5-Nme9) display a single or several different NDK domains, associated or not associated with extra-domains. Data strongly suggest that, unlike Group I, none of the members of Group II display measurable NDPK activity, although some of them autophosphorylate. The multimeric form is required for the NDPK activity. Group I proteins are known to multimerize, while there are no data on the multimerization of Group II proteins. The Group II ancestral type protein was shown to be conserved in several species from three eukaryotic supergroups. Here, we analysed the Nme protein from an early branching eukaryotic lineage, the red alga Chondrus crispus. We show that the ancestral type protein, unlike its human homologue, was fully functional multimeric NDPK with high affinity to various types of DNA and dispersed localization throughout the eukaryotic cell. Its overexpression inhibits both cell proliferation and the anchorage-independent growth of cells in soft agar but fails to deregulate cell apoptosis. We conclude that the ancestral gene has changed during eukaryotic evolution, possibly in correlation with the protein function.

Comparative genomic analyses of transport proteins encoded within the red algae Chondrus crispus, Galdieria sulphuraria, and Cyanidioschyzon merolae11.

Galdieria sulphuraria and Cyanidioschyzon merolae are thermo-acidophilic unicellular red algal cousins capable of living in volcanic environments, although the former can additionally thrive in the presence of toxic heavy metals. Bioinformatic analyses of transport systems were carried out on their genomes, as well as that of the mesophilic multicellular red alga Chondrus crispus (Irish moss). We identified transport proteins related to the metabolic capabilities, physiological properties, and environmental adaptations of these organisms. Of note is the vast array of transporters encoded in G. sulphuraria capable of importing a variety of carbon sources, particularly sugars and amino acids, while C. merolae and C. crispus have relatively few such proteins. Chondrus crispus may prefer short chain acids to sugars and amino acids. In addition, the number of encoded proteins pertaining to heavy metal ion transport is highest in G. sulphuraria and lowest in C. crispus. All three organisms preferentially utilize secondary carriers over primary active transporters, suggesting that their primary source of energy derives from electron flow rather than substrate-level phosphorylation. Surprisingly, the percentage of inorganic ion transporters encoded in C. merolae more closely resembles that of C. crispus than G. sulphuraria, but only C. crispus appears to signal via voltage-gated cation channels and possess a Na+ /K+ -ATPase and a Na+ exporting pyrophosphatase. The results presented in this report further our understanding of the metabolic potential and toxic compound resistances of these three organisms.

Identification and Characterization of miRNAs in Chondrus crispus by High-Throughput Sequencing and Bioinformatics Analysis.

Chondrus crispus, an economically and medicinally important red alga, is a medicinally active substance and important for anti-tumor research. In this study, 117 C. crispus miRNAs (108 conserved and 9 novel) were identified from 2,416,181 small-RNA reads using high-throughput sequencing and bioinformatics methods. According to the BLAST search against the miRBase database, these miRNAs belonged to 110 miRNA families. Sequence alignment combined with homology searching revealed both the conservation and diversity of predicted potential miRNA families in different plant species. Four and 19 randomly selected miRNAs were validated by northern blotting and stem-loop quantitative real-time reverse transcription polymerase chain reaction detection, respectively. The validation rates (75% and 94.7%) demonstrated that most of the identified miRNAs could be credible. A total of 160 potential target genes were predicted and functionally annotated by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. We also analyzed the interrelationship of miRNAs, miRNA-target genes and target genes in C. crispus by constructing a Cytoscape network. The 117 miRNAs identified in our study should supply large quantities of information that will be important for red algae small RNA research.

Mutant swarms of a totivirus-like entities are present in the red macroalga Chondrus crispus and have been partially transferred to the nuclear genome.

Chondrus crispus Stackhouse (Gigartinales) is a red seaweed found on North Atlantic rocky shores. Electrophoresis of RNA extracts showed a prominent band with a size of around 6,000 bp. Sequencing of the band revealed several sequences with similarity to totiviruses, double-stranded RNA viruses that normally infect fungi. This virus-like entity was named C. crispus virus (CcV). It should probably be regarded as an extreme viral quasispecies or a mutant swarm since low identity (<65%) was found between sequences. Totiviruses typically code for two genes: one capsid gene (gag) and one RNA-dependent RNA polymerase gene (pol) with a pseudoknot structure between the genes. Both the genes and the intergenic structures were found in the CcV sequences. A nonidentical gag gene was also found in the nuclear genome of C. crispus, with associated expressed sequence tags (EST) and upstream regulatory features. The gene was presumably horizontally transferred from the virus to the alga. Similar dsRNA bands were seen in extracts from different life cycle stages of C. crispus and from all geographic locations tested. In addition, similar bands were also observed in RNA extractions from other red algae; however, the significance of this apparently widespread phenomenon is unknown. Neither phenotype caused by the infection nor any virus particles or capsid proteins were identified; thus, the presence of viral particles has not been validated. These findings increase the known host range of totiviruses to include marine red algae.

Phylogeographic structure and deep lineage diversification of the red alga Chondrus ocellatus Holmes in the Northwest Pacific.

A major goal of phylogeographic analysis using molecular markers is to understand the ecological and historical variables that influence genetic diversity within a species. Here, we used sequences of the mitochondrial Cox1 gene and nuclear internal transcribed spacer to reconstruct its phylogeography and demographic history of the intertidal red seaweed Chondrus ocellatus over most of its geographical range in the Northwest Pacific. We found three deeply separated lineages A, B and C, which diverged from one another in the early Pliocene-late Miocene (c. 4.5-7.7 Ma). The remarkably deep divergences, both within and between lineages, appear to have resulted from ancient isolations, accelerated by random drift and limited genetic exchange between regions. The disjunct distributions of lineages A and C along the coasts of Japan may reflect divergence during isolation in scattered refugia. The distribution of lineage B, from the South China Sea to the Korean Peninsula, appears to reflect postglacial recolonizations of coastal habitats. These three lineages do not coincide with the three documented morphological formae in C. ocellatus, suggesting that additional cryptic species may exist in this taxon. Our study illustrates the interaction of environmental variability and demographic processes in producing lineage diversification in an intertidal seaweed and highlights the importance of phylogeographic approaches for discovering cryptic marine biodiversity.

O father where art thou? Paternity analyses in a natural population of the haploid-diploid seaweed Chondrus crispus.

The link between life history traits and mating systems in diploid organisms has been extensively addressed in the literature, whereas the degree of selfing and/or inbreeding in natural populations of haploid-diploid organisms, in which haploid gametophytes alternate with diploid sporophytes, has been rarely measured. Dioecy has often been used as a proxy for the mating system in these organisms. Yet, dioecy does not prevent the fusion of gametes from male and female gametophytes originating from the same sporophyte. This is likely a common occurrence when spores from the same parent are dispersed in clumps and recruit together. This pattern of clumped spore dispersal has been hypothesized to explain significant heterozygote deficiency in the dioecious haploid-diploid seaweed Chondrus crispus. Fronds and cystocarps (structures in which zygotes are mitotically amplified) were sampled in two 25 m(2) plots located within a high and a low intertidal zone and genotyped at 5 polymorphic microsatellite loci in order to explore the mating system directly using paternity analyses. Multiple males sired cystocarps on each female, but only one of the 423 paternal genotypes corresponded to a field-sampled gametophyte. Nevertheless, larger kinship coefficients were detected between males siring cystocarps on the same female in comparison with males in the entire population, confirming restricted spermatial and clumped spore dispersal. Such dispersal mechanisms may be a mode of reproductive assurance due to nonmotile gametes associated with putatively reduced effects of inbreeding depression because of the free-living haploid stage in C. crispus.

RT-qPCR normalization genes in the red alga Chondrus crispus.

Chondrus crispus is a common red macroalga living on the rocky shores of the North Atlantic Ocean. It has a long research history, being a major source of carrageenan, a thickener widely used in the food industry, but also for physiological and ecological studies. To establish it as a model for red algae, its genome has been sequenced, allowing the development of molecular tools such as quantification of gene expression, including RNAseq and RT-qPCR. To determine appropriate genes for RT-qPCR normalization, the expression of 14 genes was monitored in 18 conditions using two sets of algal samples: samples from the sequenced strain, cultured and stressed in laboratory conditions and C. crispus collected on the shore and stressed in situ. The expression stability of the genes between the samples was evaluated by comparing the Ct range and using the programs geNorm and NormFinder. The candidate genes encoded translation related proteins (initiation factors IF4A-1 and IF4A-2, elongation factor EF1α and eRF3, an eukaryotic polypeptide chain release factor), cytoskeleton proteins (two ß-tubulins, α-tubulin and actin), enzymes involved in the pentose phosphate pathway (glucose 6-phosphate deshydrogenase), protein recycling process (ubiquitin and ubiquitin-conjugating enzyme) and glycolysis (isocitrate dehydrogenase). The two sets of samples showed different expression patterns. Most of the genes were stable in the algae cultivated in the laboratory, whereas environmental samples showed a more important variation in gene expression. When analyzing the two sets separately, the ranking of the most stables genes were different from one method to another. When considering all samples, the two statistical methods were concordant, revealing translation initiation factor 4A-2 and eukaryotic polypeptide chain release factor 3 as pertinent normalization genes. This study highlights thus the importance of testing reference genes according to the experiments as well as the genetic and physiological background of the organism.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

Intergametophytic selfing and microgeographic genetic structure shape populations of the intertidal red seaweed Chondrus crispus.

Understanding how abiotic factors influence the spatial distribution of genetic variation provides insight into microevolutionary processes. The intertidal seascape is characterized by highly heterogeneous habitats which probably influence the partitioning of genetic variation at very small scales. The effects of tidal height on genetic variation in both the haploid (gametophytes) and diploid (tetrasporophytes) stages of the red alga Chondrus crispus were studied. Fronds were sampled every 25 cm within a 5 m × 5 m grid and along a 90-m transect at two shore heights (high and low) in one intertidal site in France. The multilocus genotype of 799 fronds was determined (Nhaploid  = 586; Ndiploid  = 213) using eight microsatellite loci to test the following hypotheses: (i) high and low shore fronds belong to genetically differentiated populations, (ii) gene flow is restricted within the high shore habitat due to tidal-influenced isolation and (iii) significant FIS values are driven by life history characteristics. Pairwise FST estimates between high and low shore levels supported the hypothesis that high and low shore fronds were genetically differentiated. The high shore was characterized by the occurrence of within-shore genetic differentiation, reduced genetic diversity and increased levels of intergametophytic selfing, suggesting it is a marginal environment. These results suggest at fine scales within the intertidal seascape the same mechanisms as those over the species' distributional range are at work with core and marginal population dynamics.

Intertidal population genetic dynamics at a microgeographic seascape scale.

The intertidal community is among the most physically harsh niches on earth, with highly heterogeneous environmental and biological factors that impose strong habitat selection on population abundance, genetic connectivity and ecological adaptation of organisms in nature. However, most genetic studies to date have concentrated on the influence of basin-wide or regional marine environments (e.g. habitat discontinuities, oceanic currents and fronts, and geographic barriers) on spatiotemporal distribution and composition of intertidal invertebrates having planktonic stages or long-distance dispersal capability. Little is known about sessile marine organisms (e.g. seaweeds) in the context of topographic tidal gradients and reproductive traits at the microgeographic scale. In this issue of Molecular Ecology, Krueger-Hadfield et al. () implemented an elaborate sampling strategy with red seaweed (Chondrus crispus) from a 90-m transect stand near Roscoff and comprehensively detected genome-scale genetic differentiation and biases in ploidy level. This study not only revealed that tidal height resulted in genetic differentiation between high- and low-shore stands and restricted the genetic exchange within the high-shore habitat, but also demonstrated that intergametophytic nonrandom fertilization in C. crispus can cause significant deviation from Hardy-Weinberg equilibrium. Such new genetic insights highlight the importance of microgeographic genetic dynamics and life history characteristics for better understanding the evolutionary processes of speciation and diversification of intertidal marine organisms.

Unique genetic variation at a species' rear edge is under threat from global climate change.

Global climate change is having a significant effect on the distributions of a wide variety of species, causing both range shifts and population extinctions. To date, however, no consensus has emerged on how these processes will affect the range-wide genetic diversity of impacted species. It has been suggested that species that recolonized from low-latitude refugia might harbour high levels of genetic variation in rear-edge populations, and that loss of these populations could cause a disproportionately large reduction in overall genetic diversity in such taxa. In the present study, we have examined the distribution of genetic diversity across the range of the seaweed Chondrus crispus, a species that has exhibited a northward shift in its southern limit in Europe over the last 40 years. Analysis of 19 populations from both sides of the North Atlantic using mitochondrial single nucleotide polymorphisms (SNPs), sequence data from two single-copy nuclear regions and allelic variation at eight microsatellite loci revealed unique genetic variation for all marker classes in the rear-edge populations in Iberia, but not in the rear-edge populations in North America. Palaeodistribution modelling and statistical testing of alternative phylogeographic scenarios indicate that the unique genetic diversity in Iberian populations is a result not only of persistence in the region during the last glacial maximum, but also because this refugium did not contribute substantially to the recolonization of Europe after the retreat of the ice. Consequently, loss of these rear-edge populations as a result of ongoing climate change will have a major effect on the overall genetic diversity of the species, particularly in Europe, and this could compromise the adaptive potential of the species as a whole in the face of future global warming.

Post-pleistocene demographic history of the North Atlantic endemic Irish moss Chondrus crispus: glacial survival, spatial expansion and gene flow.

Range expansions and gene flow as micro-evolutionary processes played a leading role in the population demographic history of marine organisms. Herein, we sequenced partial mtDNA Cox1 gene from 26 assigned geographical populations to understand how Irish moss (Chondrus crispus) responded to severe climatic oscillations during the Pleistocene glaciations and contemporary forces such as gene flow. Phylogeographic patterns indicated that haplotype frequency distributions were strongly skewed, with nearly half found only in single samples and thus restricted to a single population. Analysis of molecular variance revealed that most of the variation was within populations with no significant genetic structuring on either side of the Atlantic. Demographic analyses indicated that ISI (Irish Sea and Ireland) and NS (the North Sea) areas experienced a slight trend of increase in population size over time, whereas EC (the English Channel) area experienced expansion beginning approximately 170,000-360,000 BP. The observed complex genetic pattern of C. crispus is consistent with a scenario of multiple unrelated founding events by survival of this species in at least three putative Pleistocene refugia along the European coastline, and subsequent trans-Atlantic dispersal combined with contiguous northward population expansion predating the LGM and geographically gene flow.

Truncation of subunit ND2 disrupts the threefold symmetry of the antiporter-like subunits in complex I from higher metazoans.

Three of the conserved, membrane-bound subunits in NADH:ubiquinone oxidoreductase (complex I) are related to one another, and to Mrp sodium-proton antiporters. Recent structural analysis of two prokaryotic complexes I revealed that the three subunits each contain fourteen transmembrane helices that overlay in structural alignments: the translocation of three protons may be coordinated by a lateral helix connecting them together (Efremov, R.G., Baradaran, R. and Sazanov, L.A. (2010). The architecture of respiratory complex I. Nature 465, 441-447). Here, we show that in higher metazoans the threefold symmetry is broken by the loss of three helices from subunit ND2; possible implications for the mechanism of proton translocation are discussed.

Response of the transcriptome of the intertidal red seaweed Chondrus crispus to controlled and natural stresses.

Intertidal seaweeds inhabit an inherently stressful environment with rapidly changing physical conditions with the turning tides. Many macroalgae are therefore very resistant to abiotic stress; however, the bases for this tolerance and the relative importance of different stressors are largely unknown. Here, the effects of stress on the transcriptome of the red seaweed Chondrus crispus were investigated using cDNA microarrays. The responses were studied after exposure to high light, high temperature, and hypo- and hyperosmotic conditions in the laboratory and compared with gene expression in nature at different stress loads: at high and low tide at solar noon, and during a cloudy and a sunny day, respectively. The study identifies key stress genes and marker genes for specific stressors. The data also provide an insight into the physiological effects of stress; for example, high light stress and high natural stress caused an increase in antioxidative proteins, suggesting an increased oxidative stress. Clustering analysis suggested that osmotic stress modulated the gene expression in nature under high-stress conditions and was thus the most significant natural stressor. The potential cross-talk between stress reactions and methyl jasmonate-induced responses was also investigated and is tentatively suggested to be mediated by reactive oxygen species.

Expression profiling of Chondrus crispus (Rhodophyta) after exposure to methyl jasmonate.

Methyl jasmonate (MeJA) is a plant hormone important for the mediation of signals for developmental processes and defence reactions in higher plants. The effects of MeJA and the signalling pathways on other photosynthetic organism groups are largely unknown, even though MeJA may have very important roles. Therefore the effects of MeJA in a red alga were studied. A medium-scale expression profiling approach to identify genes regulated by MeJA in the red seaweed Chondrus crispus is described here. The expression profiles were studied 0, 2, 4, 6, 12, and 24 h after the addition of MeJA to the seawater surrounding the algae. The changes in the transcriptome were monitored using cDNA microarrays with 1920 different cDNA representing 1295 unique genes. The responses of selected genes were verified with real-time PCR and the correlation between the two methods was generally satisfying. The study showed that 6% of genes studied showed a response to the addition of MeJA and the most dynamic response was seen after 6 h. Genes that showed up-regulation included several glutathione S-transferases, heat shock protein 20, a xenobiotic reductase, and phycocyanin lyase. Down-regulated transcripts included glucose kinase, phosphoglucose isomerase, and a ribosomal protein. A comparison between different functional groups showed an up-regulation of stress-related genes and a down-regulation of genes involved in energy conversion and general metabolism. It is concluded that MeJA, or a related compound, has a physiological role as a stress hormone in red algae. This study represents to our knowledge the first analysis of gene expression using cDNA microarrays in a red macroalga.