Inferring and comparing metabolism across heterogeneous sets of annotated genomes using AuCoMe.

Comparative analysis of genome-scale metabolic networks (GSMNs) may yield important information on the biology, evolution, and adaptation of species. However, it is impeded by the high heterogeneity of the quality and completeness of structural and functional genome annotations, which may bias the results of such comparisons. To address this issue, we developed AuCoMe, a pipeline to automatically reconstruct homogeneous GSMNs from a heterogeneous set of annotated genomes without discarding available manual annotations. We tested AuCoMe with three data sets, one bacterial, one fungal, and one algal, and showed that it successfully reduces technical biases while capturing the metabolic specificities of each organism. Our results also point out shared and divergent metabolic traits among evolutionarily distant algae, underlining the potential of AuCoMe to accelerate the broad exploration of metabolic evolution across the tree of life.

Insights into the potential for mutualistic and harmful host-microbe interactions affecting brown alga freshwater acclimation.

Microbes can modify their hosts' stress tolerance, thus potentially enhancing their ecological range. An example of such interactions is Ectocarpus subulatus, one of the few freshwater-tolerant brown algae. This tolerance is partially due to its (un)cultivated microbiome. We investigated this phenomenon by modifying the microbiome of laboratory-grown E. subulatus using mild antibiotic treatments, which affected its ability to grow in low salinity. Low salinity acclimation of these algal-bacterial associations was then compared. Salinity significantly impacted bacterial and viral gene expression, albeit in different ways across algal-bacterial communities. In contrast, gene expression of the host and metabolite profiles were affected almost exclusively in the freshwater-intolerant algal-bacterial communities. We found no evidence of bacterial protein production that would directly improve algal stress tolerance. However, vitamin K synthesis is one possible bacterial service missing specifically in freshwater-intolerant cultures in low salinity. In this condition, we also observed a relative increase in bacterial transcriptomic activity and the induction of microbial genes involved in the biosynthesis of the autoinducer AI-1, a quorum-sensing regulator. This could have resulted in dysbiosis by causing a shift in bacterial behaviour in the intolerant algal-bacterial community. Together, these results provide two promising hypotheses to be examined by future targeted experiments. Although they apply only to the specific study system, they offer an example of how bacteria may impact their host's stress response.

Using automated reasoning to explore the metabolism of unconventional organisms: a first step to explore host-microbial interactions.

Systems modelled in the context of molecular and cellular biology are difficult to represent with a single calibrated numerical model. Flux optimisation hypotheses have shown tremendous promise to accurately predict bacterial metabolism but they require a precise understanding of metabolic reactions occurring in the considered species. Unfortunately, this information may not be available for more complex organisms or non-cultured microorganisms such as those evidenced in microbiomes with metagenomic techniques. In both cases, flux optimisation techniques may not be applicable to elucidate systems functioning. In this context, we describe how automatic reasoning allows relevant features of an unconventional biological system to be identified despite a lack of data. A particular focus is put on the use of Answer Set Programming, a logic programming paradigm with combinatorial optimisation functionalities. We describe its usage to over-approximate metabolic responses of biological systems and solve gap-filling problems. In this review, we compare steady-states and Boolean abstractions of metabolic models and illustrate their complementarity via applications to the metabolic analysis of macro-algae. Ongoing applications of this formalism explore the emerging field of systems ecology, notably elucidating interactions between a consortium of microbes and a host organism. As the first step in this field, we will illustrate how the reduction in microbiotas according to expected metabolic phenotypes can be addressed with gap-filling problems.

Revisiting Australian Ectocarpus subulatus (Phaeophyceae) From the Hopkins River: Distribution, Abiotic Environment, and Associated Microbiota.

In 1995 a strain of Ectocarpus was isolated from Hopkins River Falls, Victoria, Australia, constituting one of few available freshwater or nearly freshwater brown algae, and the only one belonging to the genus Ectocarpus. It has since been used as a model to study acclimation and adaptation to low salinities and the role of its microbiota in these processes. To provide more background information on this model, we assessed if Ectocarpus was still present in the Hopkins river 22 years after the original finding, estimated its present distribution, described its abiotic environment, and determined its in situ microbial composition. We sampled for Ectocarpus at 15 sites along the Hopkins River as well as 10 neighboring sites and found individuals with ITS and cox1 sequences identical to the original isolate at three sites upstream of Hopkins River Falls. The salinity of the water at these sites ranged from 3.1 to 6.9, and it was rich in sulfate (1-5 mM). The diversity of bacteria associated with the algae in situ (1312 operational taxonomic units) was one order of magnitude higher than in previous studies of the original laboratory culture, and 95 alga-associated bacterial strains were isolated from algal filaments on site. In particular, species of Planctomycetes were abundant in situ but rare in laboratory cultures. Our results confirmed that Ectocarpus was still present in the Hopkins River, and the newly isolated algal and bacterial strains offer new possibilities to study the adaptation of Ectocarpus to low salinity and its interactions with its microbiome.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Meneco, a Topology-Based Gap-Filling Tool Applicable to Degraded Genome-Wide Metabolic Networks.

Increasing amounts of sequence data are becoming available for a wide range of non-model organisms. Investigating and modelling the metabolic behaviour of those organisms is highly relevant to understand their biology and ecology. As sequences are often incomplete and poorly annotated, draft networks of their metabolism largely suffer from incompleteness. Appropriate gap-filling methods to identify and add missing reactions are therefore required to address this issue. However, current tools rely on phenotypic or taxonomic information, or are very sensitive to the stoichiometric balance of metabolic reactions, especially concerning the co-factors. This type of information is often not available or at least prone to errors for newly-explored organisms. Here we introduce Meneco, a tool dedicated to the topological gap-filling of genome-scale draft metabolic networks. Meneco reformulates gap-filling as a qualitative combinatorial optimization problem, omitting constraints raised by the stoichiometry of a metabolic network considered in other methods, and solves this problem using Answer Set Programming. Run on several artificial test sets gathering 10,800 degraded Escherichia coli networks Meneco was able to efficiently identify essential reactions missing in networks at high degradation rates, outperforming the stoichiometry-based tools in scalability. To demonstrate the utility of Meneco we applied it to two case studies. Its application to recent metabolic networks reconstructed for the brown algal model Ectocarpus siliculosus and an associated bacterium Candidatus Phaeomarinobacter ectocarpi revealed several candidate metabolic pathways for algal-bacterial interactions. Then Meneco was used to reconstruct, from transcriptomic and metabolomic data, the first metabolic network for the microalga Euglena mutabilis. These two case studies show that Meneco is a versatile tool to complete draft genome-scale metabolic networks produced from heterogeneous data, and to suggest relevant reactions that explain the metabolic capacity of a biological system.

Transitions between marine and freshwater environments provide new clues about the origins of multicellular plants and algae.

Marine-freshwater and freshwater-marine transitions have been key events in the evolution of life, and most major groups of organisms have independently undergone such events at least once in their history. Here, we first compile an inventory of bidirectional freshwater and marine transitions in multicellular photosynthetic eukaryotes. While green and red algae have mastered multiple transitions in both directions, brown algae have colonized freshwater on a maximum of six known occasions, and angiosperms have made the transition to marine environments only two or three times. Next, we review the early evolutionary events leading to the colonization of current habitats. It is commonly assumed that the conquest of land proceeded in a sequence from marine to freshwater habitats. However, recent evidence suggests that early photosynthetic eukaryotes may have arisen in subaerial or freshwater environments and only later colonized marine environments as hypersaline oceans were diluted to the contemporary level. Although this hypothesis remains speculative, it is important to keep these alternative scenarios in mind when interpreting the current habitat distribution of plants and algae. Finally, we discuss the roles of structural and functional adaptations of the cell wall, reactive oxygen species scavengers, osmoregulation, and reproduction. These are central for acclimatization to freshwater or to marine environments. We observe that successful transitions appear to have occurred more frequently in morphologically simple forms and conclude that, in addition to physiological studies of euryhaline species, comparative studies of closely related species fully adapted to one or the other environment are necessary to better understand the adaptive processes.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

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.

The genome-scale metabolic network of Ectocarpus siliculosus (EctoGEM): a resource to study brown algal physiology and beyond.

Brown algae (stramenopiles) are key players in intertidal ecosystems, and represent a source of biomass with several industrial applications. Ectocarpus siliculosus is a model to study the biology of these organisms. Its genome has been sequenced and a number of post-genomic tools have been implemented. Based on this knowledge, we report the reconstruction and analysis of a genome-scale metabolic network for E. siliculosus, EctoGEM (http://ectogem.irisa.fr). This atlas of metabolic pathways consists of 1866 reactions and 2020 metabolites, and its construction was performed by means of an integrative computational approach for identifying metabolic pathways, gap filling and manual refinement. The capability of the network to produce biomass was validated by flux balance analysis. EctoGEM enabled the reannotation of 56 genes within the E. siliculosus genome, and shed light on the evolution of metabolic processes. For example, E. siliculosus has the potential to produce phenylalanine and tyrosine from prephenate and arogenate, but does not possess a phenylalanine hydroxylase, as is found in other stramenopiles. It also possesses the complete eukaryote molybdenum co-factor biosynthesis pathway, as well as a second molybdopterin synthase that was most likely acquired via horizontal gene transfer from cyanobacteria by a common ancestor of stramenopiles. EctoGEM represents an evolving community resource to gain deeper understanding of the biology of brown algae and the diversification of physiological processes. The integrative computational method applied for its reconstruction will be valuable to set up similar approaches for other organisms distant from biological benchmark models.

Transcriptomic and metabolomic analysis of copper stress acclimation in Ectocarpus siliculosus highlights signaling and tolerance mechanisms in brown algae.

BACKGROUND: Brown algae are sessile macro-organisms of great ecological relevance in coastal ecosystems. They evolved independently from land plants and other multicellular lineages, and therefore hold several original ontogenic and metabolic features. Most brown algae grow along the coastal zone where they face frequent environmental changes, including exposure to toxic levels of heavy metals such as copper (Cu). RESULTS: We carried out large-scale transcriptomic and metabolomic analyses to decipher the short-term acclimation of the brown algal model E. siliculosus to Cu stress, and compared these data to results known for other abiotic stressors. This comparison demonstrates that Cu induces oxidative stress in E. siliculosus as illustrated by the transcriptomic overlap between Cu and H2O2 treatments. The common response to Cu and H2O2 consisted in the activation of the oxylipin and the repression of inositol signaling pathways, together with the regulation of genes coding for several transcription-associated proteins. Concomitantly, Cu stress specifically activated a set of genes coding for orthologs of ABC transporters, a P1B-type ATPase, ROS detoxification systems such as a vanadium-dependent bromoperoxidase, and induced an increase of free fatty acid contents. Finally we observed, as a common abiotic stress mechanism, the activation of autophagic processes on one hand and the repression of genes involved in nitrogen assimilation on the other hand. CONCLUSIONS: Comparisons with data from green plants indicate that some processes involved in Cu and oxidative stress response are conserved across these two distant lineages. At the same time the high number of yet uncharacterized brown alga-specific genes induced in response to copper stress underlines the potential to discover new components and molecular interactions unique to these organisms. Of particular interest for future research is the potential cross-talk between reactive oxygen species (ROS)-, myo-inositol-, and oxylipin signaling.

A metabolic approach to study algal-bacterial interactions in changing environments.

Increasing evidence exists that bacterial communities interact with and shape the biology of algae and that their evolutionary histories are connected. Despite these findings, physiological studies were and still are generally carried out with axenic or at least antibiotic-treated cultures. Here, we argue that considering interactions between algae and associated bacteria is key to understanding their biology and evolution. To deal with the complexity of the resulting 'holobiont' system, a metabolism-centred approach that uses combined metabolic models for algae and associated bacteria is proposed. We believe that these models will be valuable tools both to study algal-bacterial interactions and to elucidate processes important for the acclimation of the holobiont to environmental changes.

Towards deciphering dynamic changes and evolutionary mechanisms involved in the adaptation to low salinities in Ectocarpus (brown algae).

Colonizations of freshwater by marine species are rare events, and little information is known about the underlying mechanisms. Brown algae are an independent lineage of photosynthetic and multicellular organisms from which few species inhabit freshwater. As a marine alga that is also found in freshwater, Ectocarpus is of particular interest for studying the transition between these habitats. To gain insights into mechanisms of the transition, we examined salinity tolerance and adaptations to low salinities in a freshwater strain of Ectocarpus on physiological and molecular levels. We show that this isolate belongs to a widely distributed and highly stress-resistant clade, and differed from the genome-sequenced marine strain in its tolerance of low salinities. It also exhibited profound, but reversible, morphological, physiological, and transcriptomic changes when transferred to seawater. Although gene expression profiles were similar in both strains under identical conditions, metabolite and ion profiles differed strongly, the freshwater strain exhibiting e.g. higher cellular contents of amino acids and nitrate, higher contents of n-3 fatty acids, and lower intracellular mannitol and sodium concentrations. Moreover, several stress markers were noted in the freshwater isolate in seawater. This finding suggests that, while high stress tolerance and plasticity may be prerequisites for the colonization of freshwater, genomic alterations have occurred that produced permanent changes in the metabolite profiles to stabilize the transition.

Maintaining beneficial alga-associated bacterial communities under heat stress: insights from controlled co-culture experiments using antibiotic-resistant bacterial strains.

Brown algae, like many eukaryotes, possess diverse microbial communities. Ectocarpus-a model brown alga-relies on these communities for essential processes, such as growth development. Controlled laboratory systems are needed for functional studies of these algal-bacterial interactions. We selected bacterial strains based on their metabolic networks to provide optimal completion of the algal metabolism, rendered them resistant to two antibiotics, and inoculate them to establish controlled co-cultures with Ectocarpus under continuous antibiotic treatment. We then monitored the stability of the resulting associations under control conditions and heat stress using 16S metabarcoding. Antibiotics strongly reduced bacterial diversity both in terms of taxonomy and predicted metabolic functions. In the inoculated sample, 63%-69% of reads corresponded to the inoculated strains, and the communities remained stable during temperature stress. They also partially restored the predicted metabolic functions of the natural community. Overall, the development of antibiotic-resistant helper cultures offers a promising route to fully controlled laboratory experiments with algae and microbiota and thus represents an important step towards generating experimental evidence for specific host-microbe interactions in the systems studied. Further work will be required to achieve full control and progressively expand our repertoire of helper strains including those currently 'unculturable'.

The Rhodoexplorer Platform for Red Algal Genomics and Whole-Genome Assemblies for Several Gracilaria Species.

Macroalgal (seaweed) genomic resources are generally lacking as compared with other eukaryotic taxa, and this is particularly true in the red algae (Rhodophyta). Understanding red algal genomes is critical to understanding eukaryotic evolution given that red algal genes are spread across eukaryotic lineages from secondary endosymbiosis and red algae diverged early in the Archaeplastids. The Gracilariales is a highly diverse and widely distributed order including species that can serve as ecosystem engineers in intertidal habitats and several notorious introduced species. The genus Gracilaria is cultivated worldwide, in part for its production of agar and other bioactive compounds with downstream pharmaceutical and industrial applications. This genus is also emerging as a model for algal evolutionary ecology. Here, we report new whole-genome assemblies for two species (Gracilaria chilensis and Gracilaria gracilis), a draft genome assembly of Gracilaria caudata, and genome annotation of the previously published Gracilaria vermiculophylla genome. To facilitate accessibility and comparative analysis, we integrated these data in a newly created web-based portal dedicated to red algal genomics (https://rhodoexplorer.sb-roscoff.fr). These genomes will provide a resource for understanding algal biology and, more broadly, eukaryotic evolution.

Effects of sampling and storage procedures on 16S rDNA amplicon sequencing results of kelp microbiomes.

Brown macroalgae, including the kelp Saccharina latissima, are of both ecological and increasing economic interest. Together with their microbiota, these organisms form a singular entity, the holobiont. Sampling campaigns are required to study the microbiome of algae in natural populations, but freezing samples in liquid nitrogen is complex in the field, particularly at remote locations. Here we tested two simple alternative methods for sampling the microbial diversity associated with the kelp S. latissima: silica gel conservation of tissue and swab samples preserved in DNA/RNA shield solution. We used these techniques to compare apex and meristem samples from Roscoff (Brittany, France) and evaluated their impact on the results of 16S rDNA metabarcoding experiments. Both methods were able to separate apex and meristem microbiomes, and the results were concordant with results obtained for flash-frozen samples. However, differences were observed for several rare genera and ASVs, and the detection of contaminant sequences in the silica gel-preserved samples underline the importance of including blank samples for this method. Globally, our results confirm that the silica gel technique and swabbing combined with DNA/RNA shield preservation are valid alternatives to liquid nitrogen preservation when sampling brown macroalgae in the field. However, they also underline that, regardless of the method, caution should be taken when interpreting data on rare sequences.

The Saccharina latissima microbiome: Effects of region, season, and physiology.

Introduction: Saccharina latissima is a canopy-forming species of brown algae and, as such, is considered an ecosystem engineer. Several populations of this alga are exploited worldwide, and a decrease in the abundance of S. latissima at its southern distributional range limits has been observed. Despite its economic and ecological interest, only a few data are available on the composition of microbiota associated with S. latissima and its role in algal physiologyn. Methods: We studied the whole bacterial community composition associated with S. latissima samples from three locations (Brittany, Helgoland, and Skagerrak) by 16S metabarcoding analyses at different scales: algal blade part, regions, season (at one site), and algal physiologic state. Results and Discussion: We have shown that the difference in bacterial composition is driven by factors of decreasing importance: (i) the algal tissues (apex/meristem), (ii) the geographical area, (iii) the seasons (at the Roscoff site), and (iv) the algal host's condition (healthy vs. symptoms). Overall, Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia dominated the general bacterial communities. Almost all individuals hosted bacteria of the genus Granulosicoccus, accounting for 12% of the total sequences, and eight additional core genera were identified. Our results also highlight a microbial signature characteristic for algae in poor health independent of the disease symptoms. Thus, our study provides a comprehensive overview of the S. latissima microbiome, forming a basis for understanding holobiont functioning.

Microarray estimation of genomic inter-strain variability in the genus Ectocarpus (Phaeophyceae).

BACKGROUND: Brown algae of the genus Ectocarpus exhibit high levels of genetic diversity and variability in morphological and physiological characteristics. With the establishment of E. siliculosus as a model and the availability of a complete genome sequence, it is now of interest to analyze variability among different species, ecotypes, and strains of the genus Ectocarpus both at the genome and the transcriptome level. RESULTS: We used an E. siliculosus gene expression microarray based on EST sequences from the genome-sequenced strain (reference strain) to carry out comparative genome hybridizations for five Ectocarpus strains: four E. siliculosus isolates (the male genome strain, a female strain used for outcrosses with the genome strain, a strain isolated from freshwater, and a highly copper-tolerant strain), as well as one strain of the sister species E. fasciculatus. Our results revealed significant genomic differences between ecotypes of the same species, and enable the selection of conserved probes for future microarray experiments with these strains. In the two closely related strains (a male and a female strain used for crosses), genomic differences were also detected, but concentrated in two smaller genomic regions, one of which corresponds to a viral insertion site. CONCLUSION: The high variability between strains supports the concept of E. siliculosus as a complex of cryptic species. Moreover, our data suggest that several parts of the Ectocarpus genome may have evolved at different rates: high variability was detected particularly in transposable elements and fucoxanthin chlorophyll a/c binding proteins.

Mannitol-1-phosphate dehydrogenase activity in Ectocarpus siliculosus, a key role for mannitol synthesis in brown algae.

Mannitol represents a major end product of photosynthesis in brown algae (Phaeophyceae), and is, with the ß-1,3-glucan laminarin, the main form of carbon storage for these organisms. Despite its importance, little is known about the genes and enzymes responsible for the metabolism of mannitol in these seaweeds. Taking benefit of the sequencing of the Ectocarpus siliculosus genome, we focussed our attention on the first step of the synthesis of mannitol (reduction of the photo-assimilate fructose-6-phosphate), catalysed by the mannitol-1-phosphate dehydrogenase (M1PDH). This activity was measured in algal extracts, and was shown to be regulated by NaCl concentration in the reaction medium. Genomic analysis revealed the presence of three putative M1PDH genes (named EsM1PHD1, EsM1PDH2 and EsM1PDH3). Sequence comparison with orthologs demonstrates the modular architecture of EsM1PHD1 and EsM1PDH2, with an additional N-terminal domain of unknown function. In addition, gene expression experiments carried out on samples harvested through the diurnal cycle, and after several short-term saline and oxidative stress treatments, showed that EsM1PDH1 is the most highly expressed of these genes, whatever the conditions tested. In order to assess the activity of the corresponding protein, this gene was expressed in Escherichia coli. Cell-free extracts prepared from bacteria containing EsM1PDH1 displayed higher M1PDH activity than bacteria transformed with an empty plasmid. Further characterisation of recombinant EsM1PDH1 activity revealed its very narrow substrate specificity, salt regulation, and sensitivity towards an inhibitor of SH-enzymes.

Integrative analysis of metabolite and transcript abundance during the short-term response to saline and oxidative stress in the brown alga Ectocarpus siliculosus.

The model brown alga Ectocarpus siliculosus undergoes extensive transcriptomic changes in response to abiotic stress, many of them related to primary metabolism and particularly to amino acid biosynthesis and degradation. In this study we seek to improve our knowledge of the mechanisms underlying the stress tolerance of this alga, in particular with regard to compatible osmolytes, by examining the effects of these changes on metabolite concentrations. We performed extensive metabolic profiling (urea, amino acids, sugars, polyols, organic acids, fatty acids) of Ectocarpus samples subjected to short-term hyposaline, hypersaline and oxidative stress, and integrated the results with previously published transcriptomic data. The most pronounced changes in metabolite concentrations occurred under hypersaline stress: both mannitol and proline were accumulated, but their low final concentrations indicate that, in this stress condition, both compounds are not likely to significantly contribute to osmoregulation at the level of the entire cell. Urea and trehalose were not detected in any of our samples. We also observed a shift in fatty acid composition from n-3 to n-6 fatty acids under high salinities, and demonstrated the salt stress-induced accumulation of small amounts of γ-aminobutyric acid (GABA). GABA could be synthesized in E. siliculosus through a salt stress-induced putrescine-degradation pathway.