Non-photosynthetic predators are sister to red algae.

Rhodophyta (red algae) is one of three lineages of Archaeplastida1, a supergroup that is united by the primary endosymbiotic origin of plastids in eukaryotes2,3. Red algae are a diverse and species-rich group, members of which are typically photoautotrophic, but are united by a number of highly derived characteristics: they have relatively small intron-poor genomes, reduced metabolism and lack cytoskeletal structures that are associated with motility, flagella and centrioles. This suggests that marked gene loss occurred around their origin4; however, this is difficult to reconstruct because they differ so much from the other archaeplastid lineages, and the relationships between these lineages are unclear. Here we describe the novel eukaryotic phylum Rhodelphidia and, using phylogenomics, demonstrate that it is a closely related sister to red algae. However, the characteristics of the two Rhodelphis species described here are nearly opposite to those that define red algae: they are non-photosynthetic, flagellate predators with gene-rich genomes, along with a relic genome-lacking primary plastid that probably participates in haem synthesis. Overall, these findings alter our views of the origins of Rhodophyta, and Archaeplastida evolution as a whole, as they indicate that mixotrophic feeding-that is, a combination of predation and phototrophy-persisted well into the evolution of the group.

The Unicellular Red Alga Cyanidioschyzon merolae-The Simplest Model of a Photosynthetic Eukaryote.

Several species of unicellular eukaryotic algae exhibit relatively simple genomic and cellular architecture. Laboratory cultures of these algae grow faster than plants and often provide homogeneous cellular populations exposed to an almost equal environment. These characteristics are ideal for conducting experiments at the cellular and subcellular levels. Many microalgal lineages have recently become genetically tractable, which have started to evoke new streams of studies. Among such algae, the unicellular red alga Cyanidioschyzon merolae is the simplest organism; it possesses the minimum number of membranous organelles, only 4,775 protein-coding genes in the nucleus, and its cell cycle progression can be highly synchronized with the diel cycle. These properties facilitate diverse omics analyses of cellular proliferation and structural analyses of the intracellular relationship among organelles. C. merolae cells lack a rigid cell wall and are thus relatively easily disrupted, facilitating biochemical analyses. Multiple chromosomal loci can be edited by highly efficient homologous recombination. The procedures for the inducible/repressive expression of a transgene or an endogenous gene in the nucleus and for chloroplast genome modification have also been developed. Here, we summarize the features and experimental techniques of C. merolae and provide examples of studies using this alga. From these studies, it is clear that C. merolae-either alone or in comparative and combinatory studies with other photosynthetic organisms-can provide significant insights into the biology of photosynthetic eukaryotes.

Synergising biomass growth kinetics and transport mechanisms to simulate light/dark cycle effects on photo-production systems.

Light attenuation is a primary challenge limiting the upscaling of photobioreactors for sustainable bio-production. One key to this challenge, is to model and optimise the light/dark cycles so that cells within the dark region can be frequently transferred to the light region for photosynthesis. Therefore, this study proposes the first mechanistic model to integrate the light/dark cycle effects into biomass growth kinetics. This model was initially constructed through theoretical derivation based on the intracellular reaction kinetics, and was subsequently modified by embedding a new parameter, effective light coefficient, to account for the effects of culture mixing. To generate in silico process data, a new multiscale reactive transport modelling strategy was developed to couple fluid dynamics with biomass growth kinetics and light transmission. By comparing against previous experimental and computational studies, the multiscale model shows to be of high accuracy. Based on its simulation result, an original correlation was proposed to link effective light coefficient with photobioreactor gas inflow rate; this has not been done before. The impact of this study is that by using the proposed mechanistic model and correlation, we can easily control and optimise photobioreactor gas inflow rates to alleviate light attenuation and maintain a high biomass growth rate.

Terpene Biosynthesis in Red Algae Is Catalyzed by Microbial Type But Not Typical Plant Terpene Synthases.

Red algae (Rhodophyta) and land plants belong to the monophyletic clade Archaeplastida, and taxa of both groups are rich producers of terpene secondary metabolites. The terpene carbon skeletons of land plants are made by two types of terpene synthases: typical plant terpene synthases and microbial-type terpene synthases (MTPSLs); however, terpene biosynthesis in red algae is poorly understood. By systematic sequence analysis of seven genomes and 34 transcriptomes of red algae, MTPSL homologs were identified within one genome and two transcriptomes, whereas no homolog of typical plant terpene synthase genes was found. Phylogenetic analysis showed that red algae MTPSLs group with bacterial terpene synthases. Analysis of the genome assembly and characterization of neighboring genes demonstrated red algal MTPSLs to be bona fide red algal genes and not microbial contaminants. MTPSL genes from Porphyridium purpureum and Erythrolobus australicus were characterized via heterologous expression in Escherichia coli and demonstrated to have sesquiterpene synthase activities. We detected a number of volatile sesquiterpenes in the headspace of P. purpureum and E. australicus cultures, most identical to the in vitro products of the respective MTPSLs. Expression of the MTPSL gene in P. purpureum was found to be induced by methyl jasmonate, suggesting a role for this gene in host defense. In summary, this study indicates that the formation of terpene carbon skeletons in red algae is carried out by MTPSLs that are phylogenetically unrelated to typical plant terpene synthases and most likely originated in Rhodophyta via horizontal gene transfer from bacteria.

Three-dimensional preservation of cellular and subcellular structures suggests 1.6 billion-year-old crown-group red algae.

The ~1.6 Ga Tirohan Dolomite of the Lower Vindhyan in central India contains phosphatized stromatolitic microbialites. We report from there uniquely well-preserved fossils interpreted as probable crown-group rhodophytes (red algae). The filamentous form Rafatazmia chitrakootensis n. gen, n. sp. has uniserial rows of large cells and grows through diffusely distributed septation. Each cell has a centrally suspended, conspicuous rhomboidal disk interpreted as a pyrenoid. The septa between the cells have central structures that may represent pit connections and pit plugs. Another filamentous form, Denaricion mendax n. gen., n. sp., has coin-like cells reminiscent of those in large sulfur-oxidizing bacteria but much more recalcitrant than the liquid-vacuole-filled cells of the latter. There are also resemblances with oscillatoriacean cyanobacteria, although cell volumes in the latter are much smaller. The wider affinities of Denaricion are uncertain. Ramathallus lobatus n. gen., n. sp. is a lobate sessile alga with pseudoparenchymatous thallus, "cell fountains," and apical growth, suggesting florideophycean affinity. If these inferences are correct, Rafatazmia and Ramathallus represent crown-group multicellular rhodophytes, antedating the oldest previously accepted red alga in the fossil record by about 400 million years.

Dynamics of the nucleoside diphosphate kinase protein DYNAMO2 correlates with the changes in the global GTP level during the cell cycle of Cyanidioschyzon merolae.

GTP is an essential source of energy that supports a large array of cellular mechanochemical structures ranging from protein synthesis machinery to cytoskeletal apparatus for maintaining the cell cycle. However, GTP regulation during the cell cycle has been difficult to investigate because of heterogenous levels of GTP in asynchronous cell cycles and genetic redundancy of the GTP-generating enzymes. Here, in the unicellular red algae Cyanidioschyzon merolae, we demonstrated that the ATP-GTP-converting enzyme DYNAMO2 is an essential regulator of global GTP levels during the cell cycle. The cell cycle of C. merolae can be highly synchronized by light/dark stimulations to examine GTP levels at desired time points. Importantly, the genome of C. merolae encodes only two isoforms of the ATP-GTP-converting enzyme, namely DYNAMO1 and DYNAMO2. DYNAMO1 regulates organelle divisions, whereas DYNAMO2 is entirely localized in the cytoplasm. DYNAMO2 protein levels increase during the S-M phases, and changes in GTP levels are correlated with these DYNAMO2 protein levels. These results indicate that DYNAMO2 is a potential regulator of global GTP levels during the cell cycle.

Bending strategies of convergently evolved, articulated coralline algae.

The evolution of uncalcified genicula in upright calcified corallines has occurred at least three times independently, resulting in articulated corallines within Corallinoideae, Lithophylloideae, and Metagoniolithoideae. Genicula confer flexibility to otherwise rigid thalli, and the localization of bending at discrete intervals amplifies bending stress in genicular tissue. Genicular morphology must, therefore, be balanced between maintaining flexibility while mitigating or resisting stress. Genicula in the three articulated lineages differ in both cellular construction and development, which may result in different constraints on morphology. By studying the interaction between flexibility and morphological variation in multiple species, we investigate whether representatives of convergently evolving clades follow similar strategies to generate mechanically successful articulated fronds. By using computational models to explore different bending strategies, we show that there are multiple ways to generate flexibility in upright corallines but not all morphological strategies are mechanically equivalent. Corallinoids have many joints, lithophylloids have pliant joints, and metagoniolithoids have longer joints-while these strategies can lead to comparable thallus flexibility, they also lead to different levels of stress amplification in bending. Moreover, genicula at greatest risk of stress amplification are typically the strongest, universally mitigating the trade-off between flexibility and stress reduction.

Role of abscisic acid (ABA) in activating antioxidant tolerance responses to desiccation stress in intertidal seaweed species.

MAIN CONCLUSION: The hormone ABA regulates the oxidative stress state under desiccation in seaweed species; an environmental condition generated during daily tidal changes. Desiccation is one of the most important factors that determine the distribution pattern of intertidal seaweeds. Among most tolerant seaweed is Pyropia orbicularis, which colonizes upper intertidal zones along the Chilean coast. P. orbicularis employs diverse mechanisms of desiccation tolerance (DT) (among others, e.g., antioxidant activation, photoinhibition, and osmo-compatible solute overproduction) such as those used by resurrection plants and bryophytes. In these organisms, the hormone abscisic acid (ABA) plays an important role in regulating responses to water deficit, including gene expression and the activity of antioxidant enzymes. The present study determined the effect of ABA on the activation of antioxidant responses during desiccation in P. orbicularis and in the sensitive species Mazzaella laminarioides and Lessonia spicata. Changes in endogenous free and conjugated ABA, water content during the hydration-desiccation cycle, enzymatic antioxidant activities [ascorbate peroxidase (AP), catalase (CAT) and peroxiredoxine (PRX)], and levels of lipid peroxidation and cell viability were evaluated. The results showed that P. orbicularis had free ABA levels 4-7 times higher than sensitive species, which was overproduced during water deficit. Using two ABA inhibitors (sodium tungstate and ancymidol), ABA was found to regulate the activation of the antioxidant enzymes activities during desiccation. In individuals exposed to exogenous ABA the enzyme activity increased, concomitant with low lipid peroxidation and high cell viability. These results demonstrate the participation of ABA in the regulation of DT in seaweeds, and suggest that regulatory mechanisms with ABA signaling could be of great importance for the adaptation of these organisms to dehydration.

Species diversity of the genus Osmundea (Ceramiales, Rhodophyta) in the Macaronesian region.

Species diversity within the genus Osmundea in the Macaronesian region was explored by conducting a comprehensive sampling in the Azores, the Canary, and the Madeira archipelagos. Toward identification, all specimens were first observed alive to verify the absence of corps en cerise, a diagnostic character for the genus and morphometric data were measured (thallus length and width, first-order branches length and width, branchlets length and width, cortical cell length and width in surface view, cortical cell length and width in transverse section). Specimens were sequenced for COI-5P (39 specimens) and three species delimitation methods (Generalized Mixed Yule Coalescent, Automatic Barcode Gap Discovery method, and Poisson Tree Processes) were used to assess the threshold between infra- and interspecific relationships. Subsequently, one or several sequences of plastid-encoded large subunit of RuBisCO (21 specimens) per delimited species were generated to assess the phylogenetic relationships among Macaronesian Osmundea. Moreover, for each delineated species, vegetative and reproductive anatomy was thoroughly documented and, when possible, specimens were either assigned to existing taxa or described as novel species. This integrative approach has provided data for (i) the presence of O. oederi, O. pinnatifida, and O. truncata in Macaronesia; (ii) the proposal of two novel species, O. prudhommevanreinei sp. nov. and O. silvae sp. nov.; and (iii) evidence of an additional species referred as "Osmundea sp.1," which is a sister taxon of O. hybrida.

Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae.

Effects of eutrophic seawater and temperature on the physiology and morphology of Hypnea musciformis J. V. Lamouroux (Gigartinales, Rhodophyta).

As both food and source of a kappa-carrageenan, Hypnea musciformis represents a species of great economic interest. It also synthesizes substances with antiviral, anti-helminthic and anti-inflammatory potential and shows promise for use as a bioindicator of cadmium. In this study, we investigated the combined effects of seawater from three urbanized areas (area 1: natural runoff, NRA; area 2: urbanized runoff and sewage with treatment, RTA; area 3: urbanized runoff and untreated sewage, RUS) and three different temperatures (15, 25 and 30 °C) on the growth rate, photosynthetic efficiency, photosynthetic pigments and cell morphology of H. musciformis. After 4 days (96 h) of culture, the biomass of H. musciformis showed differences that fluctuated among the areas and temperature treatments. Specifically, the specimens cultivated in 35 °C had low values of ETRmax, α(ETR), ß(ETR), and Fv/Fm photosynthetic parameters, as well as changes in cell morphology, with reduction in photosynthetic pigments and drastic reduction in growth rates. When combined with the extreme temperatures, high concentrations of ammonium ion in seawater effluent caused an inhibition of photosynthetic activity, as well as significant variation in chlorophyll a and carotenoid contents. As observed by light microscopy, the synergism between different temperatures and pollutants found in eutrophic waters caused changes in cellular morphology with increased cell wall thickening and decreased floridean starch grains. H. musciformis also showed important changes in physiological response to each factor independently, as well as changes resulting from the synergistic interaction of these factors combined. Therefore, we can conclude that extreme temperature combined with the effect of eutrophic waters, especially RUS, caused distinct morphological and physiological changes in the red alga H. musciformis.

Stable expression of a GFP-reporter gene in the red alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae is used as a model organism to investigate the basic architecture of photosynthetic eukaryotes. We established a stable expression system for the green fluorescent protein fused with the phycocyanin-associated rod linker (APCC) protein in C. merolae, which was clearly localized on the plastid. This system should be useful in the genetic engineering of C. merolae.

The effect of cadmium under different salinity conditions on the cellular architecture and metabolism in the red alga Pterocladiella capillacea (Rhodophyta, Gelidiales).

The in vitro effect of cadmium (Cd) on apical segments of Pterocladiella capillacea was examined. Over a period of 7 days, the segments were cultivated with the combination of different salinities (25, 35, and 45 practical salinity units) and Cd concentrations, ranging from 0.17 to 0.70 ppm. The effects of Cd on growth rates and content of photosynthetic pigments were analyzed. In addition, metabolic profiling was performed, and samples were processed for microscopy. Serious damage to physiological performance and ultrastructure was observed under different combinations of Cd concentrations and salinity values. Elementary infrared spectroscopy revealed toxic effects registered on growth rate, photosynthetic pigments, chloroplast, and mitochondria organization, as well as changes in lipids and carbohydrates. These alterations in physiology and ultrastructure were, however, coupled to activation of such defense mechanisms as cell wall thickness, reduction of photosynthetic harvesting complex, and flavonoid. In conclusion, P. capillacea is especially sensitive to Cd stress when intermediate concentrations of this pollutant are associated with low salinity values. Such conditions resulted in metabolic compromise, reduction of primary productivity, i.e., photosynthesis, and carbohydrate accumulation in the form of starch granules. Taken together, these findings improve our understanding of the potential impact of this metal in the natural environment.

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle.

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.

Mitochondrial localization of ferrochelatase in a red alga Cyanidioschyzon merolae.

Ferrochelatase (FECH) is an essential enzyme for the final step of heme biosynthesis. In green plants, its activity has been reported in both plastids and mitochondria. However, the precise subcellular localization of FECH remains uncertain. In this study, we analyzed the localization of FECH in the unicellular red alga, Cyanidioschyzon merolae. Immunoblot and enzyme activity analyses of subcellular fractions localized little FECH in the plastid. In addition, immunofluorescence microscopy identified that both intrinsic and hemagglutinin (HA)-tagged FECH are localized in the mitochondrion. We therefore conclude that FECH is localized in the mitochondrion in C. merolae.

Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae.

The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin.

Extensive cryptic species diversity and fine-scale endemism in the marine red alga Portieria in the Philippines.

We investigated species diversity and distribution patterns of the marine red alga Portieria in the Philippine archipelago. Species boundaries were tested based on mitochondrial, plastid and nuclear encoded loci, using a general mixed Yule-coalescent (GMYC) model-based approach and a bayesian multilocus species delimitation method. The outcome of the GMYC analysis of the mitochondrial encoded cox2-3 dataset was highly congruent with the multilocus analysis. In stark contrast with the current morphology-based assumption that the genus includes a single, widely distributed species in the Indo-West Pacific (Portieria hornemannii), DNA-based species delimitation resulted in the recognition of 21 species within the Philippines. Species distributions were found to be highly structured with most species restricted to island groups within the archipelago. These extremely narrow species ranges and high levels of intra-archipelagic endemism contrast with the wide-held belief that marine organisms generally have large geographical ranges and that endemism is at most restricted to the archipelagic level. Our results indicate that speciation in the marine environment may occur at spatial scales smaller than 100 km, comparable with some terrestrial systems. Our finding of fine-scale endemism has important consequences for marine conservation and management.

Phylogenetic substitution models for detecting heterotachy during plastid evolution.

There is widespread evidence of lineage-specific rate variation, known as heterotachy, during protein evolution. Changes in the structural and functional constraints acting on a protein can lead to heterotachy, and it is plausible that such changes, known as covarion shifts, may affect many amino acids at once. Several previous attempts to model heterotachy have used covarion models, where the sequence undergoes covarion drift, whereby each site may switch independently among a set of discrete classes having different substitution rates. However, such independent switching may not capture biologically important events where the selective forces acting on a protein affect many sites at once. We describe a new class of models that allow the rates of substitution and switching to vary among branches of a phylogenetic tree. Such models are better able to handle covarion shifts. We apply these models to a set of genes occurring in nonphotosynthetic bacteria, cyanobacteria, and the plastids of green and red algae. We find that 4/5 genes show evidence of some form of rate switching and that 3/5 genes show evidence that the relative switching rate differs among taxonomic groups. We conclude that covarion shifts may be frequent during the deep evolution of plastid genes and that our methodology may provide a powerful new tool for investigating such shifts in other systems.

[Targeting malaria parasite at the level of apicoplast: an update]. / Paludisme - Recherche de nouvelles approches thérapeutiques ciblant l'apicoplaste, un organite cellulaire d'origine algale.

In 1996, the discovery of a relic chloroplast in Plasmodium and Toxoplasma cells has strongly changed our vision of these parasites in the "Tree of Life", and has opened an unexpected new field of investigation in the search for antiparasitic treatments, including antimalarials. This review details our current understanding of the sophisticated evolution of the parasites of the Apicomplexa phylum and briefly covers a decade of research and development in drug discovery, trying to target the malaria parasite at the level of its plant-like organelle. Fifteen years after the discovery of the apicoplast and ten years after the publication of the genome of Plasmodium falciparum, it seems that we have completed a first phase of tests of available antibiotics and herbicides. In the human host, the liver phase is the only parasitic stage, for which biological functions harbored by the apicoplast, such as fatty acid biosynthesis, seem indispensable. During the erythrocytic phase, recent results have focused the attention on the processes controlling the biogenesis of the apicoplast, and one of the functions harbored by the apicoplast, i.e. the biosynthesis of isoprenoids, as major -promising targets for future treatments.

Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae.

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.