Photosynthetic Eukaryotes Research Articles

The endosymbiont of Epithemia clementina is specialized for nitrogen fixation within a photosynthetic eukaryote.

Epithemia spp. diatoms contain obligate, nitrogen-fixing endosymbionts, or diazoplasts, derived from cyanobacteria. These algae are a rare example of photosynthetic eukaryotes that have successfully coupled oxygenic photosynthesis with oxygen-sensitive nitrogenase activity. Here, we report a newly-isolated species, E. clementina, as a model to investigate endosymbiotic acquisition of nitrogen fixation. We demonstrate that the diazoplast, which has lost photosynthesis, provides fixed nitrogen to the diatom host in exchange for fixed carbon. To identify the metabolic changes associated with this endosymbiotic specialization, we compared the Epithemia diazoplast with its close, free-living cyanobacterial relative, Crocosphaera subtropica. Unlike C. subtropica, in which nitrogenase activity is temporally separated from photosynthesis, we show that nitrogenase activity in the diazoplast is continuous through the day (concurrent with host photosynthesis) and night. Host and diazoplast metabolism are tightly coupled to support nitrogenase activity: Inhibition of photosynthesis abolishes daytime nitrogenase activity, while nighttime nitrogenase activity no longer requires cyanobacterial glycogen storage pathways. Instead, import of host-derived carbohydrates supports nitrogenase activity throughout the day-night cycle. Carbohydrate metabolism is streamlined in the diazoplast compared to C. subtropica with retention of the oxidative pentose phosphate pathway and oxidative phosphorylation. Similar to heterocysts, these pathways may be optimized to support nitrogenase activity, providing reducing equivalents and ATP and consuming oxygen. Our results demonstrate that the diazoplast is specialized for endosymbiotic nitrogen fixation. Altogether, we establish a new model for studying endosymbiosis, perform a functional characterization of this diazotroph endosymbiosis, and identify metabolic adaptations for endosymbiotic acquisition of a critical biological function.

Molecular mechanisms of microbiome modulation by the eukaryotic secondary metabolite azelaic acid

Photosynthetic eukaryotes, such as microalgae and plants, foster fundamentally important relationships with their microbiome based on the reciprocal exchange of chemical currencies. Among these, the dicarboxylate metabolite azelaic acid (Aze) appears to play an important, but heterogeneous, role in modulating these microbiomes, as it is used as a carbon source for some heterotrophs but is toxic to others. However, the ability of Aze to promote or inhibit growth, as well as its uptake and assimilation mechanisms into bacterial cells are mostly unknown. Here, we use transcriptomics, transcriptional factor coexpression networks, uptake experiments, and metabolomics to unravel the uptake, catabolism, and toxicity of Aze on two microalgal-associated bacteria, Phycobacter and Alteromonas, whose growth is promoted or inhibited by Aze, respectively. We identify the first putative Aze transporter in bacteria, a ‘C4-TRAP transporter’, and show that Aze is assimilated through fatty acid degradation, with further catabolism occurring through the glyoxylate and butanoate metabolism pathways when used as a carbon source. Phycobacter took up Aze at an initial uptake rate of 3.8×10–9 nmol/cell/hr and utilized it as a carbon source in concentrations ranging from 10 μM to 1 mM, suggesting a broad range of acclimation to Aze availability. For growth-impeded bacteria, we infer that Aze inhibits the ribosome and/or protein synthesis and that a suite of efflux pumps is utilized to shuttle Aze outside the cytoplasm. We demonstrate that seawater amended with Aze becomes enriched in bacterial families that can catabolize Aze, which appears to be a different mechanism from that in soil, where modulation by the host plant is required. This study enhances our understanding of carbon cycling in the oceans and how microscale chemical interactions can structure marine microbial populations. In addition, our findings unravel the role of a key chemical currency in the modulation of eukaryote-microbiome interactions across diverse ecosystems.

Comparative analysis of organelle genomes provides conflicting evidence between morphological similarity and phylogenetic relationship in diatoms

Diatoms (Bacillariophyta) are abundant phytoplankton groups in marine environments, which contribute approximately 20% of global carbon fixation through photosynthesis. Moreover, diatoms exhibit the highest species diversity (approximately 18,000 diatom species) among marine photosynthetic eukaryotes, which were identified by morphological characteristics. Molecular phylogenetic analyses could shed new insights into the evolutionary relationships of diverse diatom species. Nevertheless, a comprehensive understanding of the phylogenetic relationships of diatom species still remains unclear because the available molecular data are insufficient compared with their high species diversity. Furthermore, several novel diatom species were reported from field samples with no molecular evidence. In particular, the phylogenies of diatom species constructed using organelle genomes revealed that several diatom genera are paraphyletic with high supporting values. We constructed high-resolution phylogenetic trees of diatom species using organelle genomes (plastids and mitochondria) and compared the morphologies in several paraphyletic diatom genera. Especially, the clades Nitzschia and Thalassiosira include several different diatom genera with high phylogenetic supports. Our study demonstrated that some morphological characteristics (e.g., genus characters) of several diatom genera could not represent current genus boundaries. Based on the results, we highlight the necessity for taxonomic reinvestigation. To reestablish this in diatoms, it will be essential to incorporate more genome data from a broader range of taxon samples, along with a comparison of morphological characteristics.

Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

Design and assembly of the 117-kb Phaeodactylum tricornutum chloroplast genome.

There is growing impetus to expand the repertoire of chassis available to synthetic biologists. Chloroplast genomes present an interesting alternative for engineering photosynthetic eukaryotes; however, development of the chloroplast as a synthetic biology chassis has been limited by a lack of efficient techniques for whole-genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117-kb Phaeodactylum tricornutum chloroplast genome that have 90% to 100% efficiency when screening as few as 10 yeast (Saccharomyces cerevisiae) colonies following yeast assembly. The first method reconstitutes the genome from PCR-amplified fragments, whereas the second method involves precloning these fragments into individual plasmids from which they can later be released. In both cases, overlapping fragments of the chloroplast genome and a cloning vector are homologously recombined into a singular contig through yeast assembly. The cloned chloroplast genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a delivery mechanism for bringing DNA directly to the algal chloroplast. Also, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 (coding for orotidine-5'-phosphate decarboxylase) open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly. The methods presented here could be extrapolated to other species-particularly those with a similar chloroplast genome size and architecture (e.g. Thalassiosira pseudonana).

A feasible method of induced biological soil crust propagation through the inoculation of moss and addition of soil amendments in a Pb-Zn tailing pond

The stacking of tailings results in serious environmental pollution and plant growth difficulty. However, moss and microorganisms can successfully colonize in tailings to form biological soil crusts (BSCs) and provide a feasible means to ecologically restore tailing reservoirs. Nonetheless, information on this approach is scarce. In this study, a 90 day field experiment was conducted to form BSCs in a Pb-Zn tailing pond in Jianshui County, China by inoculating in-situ moss crust fragments and adding three soil amendments. Results showed that induced BSCs successfully propagated, and the biomass increased to 15.51–20.33 times the initial value. Moss inoculation considerably increased the soil moisture, water-holding capacity, and phosphatase by 9.2 %, 8.8 %, and 64.0 %, respectively, and decreased exchangeable fraction Pb by 30.7 %. The co-inoculation of moss and biochar remarkably increased soil moisture, water-holding capacity, cation exchange capacity, sucrase, urease, and phosphatase activity by 22.3 %, 23.4 %, 116 %, 80.5 %, 28.6 %, and 240 %, respectively, and decreased the bulk density by 13.3 %. The addition of red soil reduced the total contents of Pb and Zn, whereas that of the stabilizer increased the pH and decreased the bioavailability of Pb and Zn. Co-inoculation greatly increased the biotic community species richness and changed their structure and function. The dominant photosynthetic eukaryotes shifted from Synechococcales to Oscillatoriales. Bacterial nutritional types shifted from chemoautotrophy to photoautotrophy and chemoautotrophy, and fungal nutritional types changed from oligotrophy to copiotrophy. These changes drove alterations in bacterial and fungal community structures. These results indicated that the propagation of induced BSCs can rapidly improve the soil structure and nutrient cycle, restore the biotic abundance and function, and facilitate the soil formation of tailings. Thus, this method holds promise for the ecological restoration of tailings.

Acquisition of Desiccation Tolerance Unveiled: Polar Lipid Profiles of Streptophyte Algae Offer Insights.

Terrestrialization by photosynthetic eukaryotes took place in the two branches of green microalgae: Chlorophyta and Charophyta. Within the latter, the paraphyletic streptophytic algae divide into two clades. These are named Klebsormidiophyceae-Chlorokybophyceae-Mesostigmatophyceae (KCM), which is the oldest, and Zygnematophyceae-Coleochaetophyceae-Charophyceae (ZCC), which contains the closest relatives of vascular plants. Terrestrialization required the emergence of adaptations in response to new challenges, such as irradiance, temperature oscillations and water deprivation. In this study, we evaluated lipid composition in species representative of distinct phylogenetic clusters within Charophyta and Chlorophyta. We aim to study whether the inherent thylakoid lipid composition, as well as its adaptability in response to desiccation, were fundamental factors for the evolutionary history of terrestrial plants. The results showed that the lipid composition was similar to that found in flowering land plants, differing only in betaine lipids. Likewise, the largest constitutive pool of oligogalactolipids (OGL) was found only in the fully desiccation-tolerant species Klebsormidium nitens. After desiccation, the content of polar lipids decreased in all species. Conversely, the content of OGL increased, particularly trigalactosyldiacylglycerol and tetragalactosyldiacylglycerol in the ZCC clade. The analysis of the molecular species composition of the newly formed OGL may suggest a different biosynthetic route for the KCM and ZCC clades. We speculate that the appearance of a new OGL synthesis pathway, which eventually arose during the streptophyte evolutionary process, endowed algae with a much more dynamic regulation of thylakoid composition in response to stress, which ultimately contributed to the colonization of terrestrial habitats.

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

Complex Plastids and the Evolution of the Marine Phytoplankton

Photosynthesis allows for the formation of biomass from inorganic carbon and therefore greatly enhances the amount of organic material on planet Earth. Especially, oxygenic photosynthesis removed a major bottleneck in the formation of biomass by utilising ubiquitous water (H2O) and CO2 molecules as raw materials for organic molecules. This, over billions of years, shaped the world into the form we know today, with an oxygen-containing atmosphere, largely oxygenated water bodies and landmasses consisting of sediment rocks. Oxygenic photosynthesis furthermore enabled the evolution of aerobic energy metabolism, and it would be very difficult to imagine animal (including human) life in the absence of molecular oxygen as an electron acceptor. Oxygenic photosynthesis first, and exclusively, evolved in cyanobacteria. However, eukaryotes also learned to photosynthesise, albeit with a trick, which is the integration of formerly free-living cyanobacteria into the eukaryotic cell. There, the former bacteria became endosymbionts, and from these endosymbionts, the photosynthetic organelles (termed plastids) evolved. In almost all major groups of eukaryotes, plastid-containing members are found. At the same time, plastid-related features also indicate that these plastids form a monophyletic group. This can be explained by the transfer of plastids between the eukaryotic super-groups, leading to plastids being found in groups that are otherwise non-photosynthetic. In this chapter, we discuss the evolutionary origin of plastids, with a special emphasis on the evolution of plankton algae, such as diatoms or dinoflagellates, who acquired their plastids from other photosynthetic eukaryotes.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A.thaliana and the single LRL/DROP protein MpLRL in M.polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A.thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M.polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum.

Phaeodactylum tricornutum plastid is surrounded by four membranes, and its protein composition and function remain mysterious. In this study, the P. tricornutum plastid-enriched fraction was obtained and 2850 proteins were identified, including 92 plastid-encoded proteins, through label-free quantitative proteomic technology. Among them, 839 nuclear-encoded proteins were further determined to be plastidial proteins based on the BLAST alignments within Plant Proteome DataBase and subcellular localization prediction, in spite of the strong contamination by mitochondria-encoded proteins and putative plasma membrane proteins. According to our proteomic data, we reconstructed the metabolic pathways and highlighted the hybrid nature of this diatom plastid. Triacylglycerol (TAG) hydrolysis and glycolysis, as well as photosynthesis, glycan metabolism, and tocopherol and triterpene biosynthesis, occur in the plastid. In addition, the synthesis of long-chain acyl-CoAs, elongation, and desaturation of fatty acids (FAs), and synthesis of lipids including TAG are confined in the four-layered-membrane plastid based on the proteomic and GFP-fusion localization data. The whole process of generation of docosahexaenoic acid (22:6) from palmitic acid (16:0), via elongation and desaturation of FAs, occurs in the chloroplast endoplasmic reticulum membrane, the outermost membrane of the plastid. Desaturation that generates 16:4 from 16:0 occurs in the plastid stroma and outer envelope membrane. Quantitative analysis of glycerolipids between whole cells and isolated plastids shows similar composition, and the FA profile of TAG was not different. This study shows that the diatom plastid combines functions usually separated in photosynthetic eukaryotes, and differs from green alga and plant chloroplasts by undertaking the whole process of lipid biosynthesis.

Johansenicoccus eremophilus gen. et sp. nov., a novel evolutionary lineage in Chlorophyceae with unusual genomic features

Background – Green algae are a diverse group of photosynthetic eukaryotes, yet are still vastly understudied compared to land plants. For many years, green algae were characterized based on their morphology and life cycles. More recently, phylogenetic and genomic analyses have been added to the phycological toolkit for a better understanding of algal biodiversity and evolutionary history. Material and methods – A desert strain of green algae was isolated from Joshua Tree National Park (JTNP) in southern California as part of a larger biodiversity survey. The alga’s nuclear rRNA genes as well as the chloroplast genome were sequenced, annotated, and analysed in addition to a morphological assessment. Results – Morphologically this strain is especially similar to Pseudomuriella and Rotundella, and its lipid profile resembles that of other soil algae, but phylogenomic analyses demonstrate that it is a distinct evolutionary lineage in Chlorophyceae. The alga exhibits several unusual genomic features, the most remarkable being its highly derived yet apparently functional nuclear rRNA genes, 18S and 28S. Both genes are GC-rich and bear many compensatory base changes to maintain a similar secondary structure to that of other green algae. The chloroplast genome has a distinct gene order and repeat arrangement from other published green algal plastomes, but contains the expected genes and also provides phylogenetically informative data. Conclusion – We conclude that the strain be placed into a new species and genus in the class Chlorophyceae, and propose the name Johansenicoccus eremophilus for this new taxon. Johansenicoccus eremophilus exemplifies science’s insufficient understanding of the range of genomic variations among inconspicuous soil algae.

Evolutionary Implications from Lipids in Membrane Bilayers and Photosynthetic Complexes in Cyanobacteria and Chloroplasts.

In biomembranes, lipids form bilayer structures that serve as the fluid matrix for membrane proteins and other hydrophobic compounds. Additionally, lipid molecules associate with membrane proteins and impact their structures and functions. In both cyanobacteria and the chloroplasts of plants and algae, the lipid bilayer of the thylakoid membrane consists of four distinct glycerolipid classes: monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol. These lipids are also integral components of photosynthetic complexes such as photosystem II and photosystem I. The lipid binding sites within the photosystems, as well as the lipid composition in the thylakoid membrane, are highly conserved between cyanobacteria and photosynthetic eukaryotes, and each lipid class has specific roles in oxygenic photosynthesis. This review aims to shed light on the potential evolutionary implications of lipid utilization in membrane lipid bilayers and photosynthetic complexes in oxygenic photosynthetic organisms.

Redox partner interactions in the ATG8 lipidation system in microalgae.

Autophagy is a catabolic pathway that functions as a degradative and recycling process to maintain cellular homeostasis in most eukaryotic cells, including photosynthetic organisms such as microalgae. This process involves the formation of double-membrane vesicles called autophagosomes, which engulf the material to be degraded and recycled in lytic compartments. Autophagy is mediated by a set of highly conserved autophagy-related (ATG) proteins that play a fundamental role in the formation of the autophagosome. The ATG8 ubiquitin-like system catalyzes the conjugation of ATG8 to the lipid phosphatidylethanolamine, an essential reaction in the autophagy process. Several studies identified the ATG8 system and other core ATG proteins in photosynthetic eukaryotes. However, how ATG8 lipidation is driven and regulated in these organisms is not fully understood yet. A detailed analysis of representative genomes from the entire microalgal lineage revealed a high conservation of ATG proteins in these organisms with the remarkable exception of red algae, which likely lost ATG genes before diversification. Here, we examine in silico the mechanisms and dynamic interactions between different components of the ATG8 lipidation system in plants and algae. Moreover, we also discuss the role of redox post-translational modifications in the regulation of ATG proteins and the activation of autophagy in these organisms by reactive oxygen species.

New record of the green algal fossil Proterocladus and coexisting microfossils from the Meso-Neoproterozoic Diaoyutai Formation in southern Liaoning, North China

The Mesoproterozoic-Neoproterozoic transition represents a crucial interval of evolutionary innovation and phylogenetic diversification. A spotty fossil record indicates that several clades of multicellular eukaryotes, including red and green algae, as well as fungi, may have diverged and diversified at this transition. However, the Meso-Neoproterozoic fossil record remains poorly documented, particularly at the assemblage level, greatly limiting our understanding of the marine ecosystem in this critical time interval. Here we report a well-preserved assemblage of organic-walled microfossils from the Meso-Neoproterozoic Diaoyutai Formation (ca. 1050–950 Ma) in Southern Liaoning Province of the North China Block. Raman geothermometric analysis of these organic-walled microfossils suggests that the Diaoyutai Formation experienced relatively low-grade metamorphism, with apparent peak metamorphic temperatures of ∼ 215 ℃. The Diaoyutai assemblage consists of 14 species belonging to 9 genera, as well as several unnamed or unidentified forms. In addition to simple forms such as aggregated coccoidal and unbranched filamentous fossils that are often interpreted as cyanobacteria, a moderate diversity of eukaryotes is also observed. Probable eukaryotic fossils in this assemblage include the branched multicellular alga Proterocladus antiquus that has been previously interpreted as a green alga, several morphologically complex spheroidal taxa (e.g., Germinosphaera bispinosa, Pterospermopsimorpha insolita, P. pileiformis and Squamosphaera colonialica), and relatively large leiospheres (50–700 μm in diameter). We note that the occurrence of Proterocladus antiquus in the Diaoyutai assemblage predates its previously reported occurrence from the overlying Nanfen Formation. Overall, the Diaoyutai assemblage presents a glimpse of the coastal marine ecosystem at the Meso-Neoproterozoic transition, which consisted of benthic microbial mats dominated by filamentous cyanobacteria, millimeter-sized benthic green algae with thallus elevating above the sediment–water interface, and micrometer-sized planktonic prokaryotic and eukaryotic organisms. From the perspective of primary producers, the rise of photosynthetic eukaryotes in the Meso-Neoproterozoic represents a transformative innovation to modernize the coastal marine ecosystem in terms of ecological functions.

An unexpected hydratase synthesizes the green light-absorbing pigment fucoxanthin.

The ketocarotenoid fucoxanthin and its derivatives can absorb blue-green light enriched in marine environments. Fucoxanthin is widely adopted by phytoplankton species as a main light-harvesting pigment, in contrast to land plants that primarily employ chlorophylls. Despite its supreme abundance in the oceans, the last steps of fucoxanthin biosynthesis have remained elusive. Here, we identified the carotenoid isomerase-like protein CRTISO5 as the diatom fucoxanthin synthase that is related to the carotenoid cis-trans isomerase CRTISO from land plants but harbors unexpected enzymatic activity. A crtiso5 knockout mutant in the model diatom Phaeodactylum tricornutum completely lacked fucoxanthin and accumulated the acetylenic carotenoid phaneroxanthin. Recombinant CRTISO5 converted phaneroxanthin into fucoxanthin in vitro by hydrating its carbon-carbon triple bond, instead of functioning as an isomerase. Molecular docking and mutational analyses revealed residues essential for this activity. Furthermore, a photophysiological characterization of the crtiso5 mutant revealed a major structural and functional role of fucoxanthin in photosynthetic pigment-protein complexes of diatoms. As CRTISO5 hydrates an internal alkyne physiologically, the enzyme has unique potential for biocatalytic applications. The discovery of CRTISO5 illustrates how neofunctionalization leads to major diversification events in evolution of photosynthetic mechanisms and the prominent brown coloration of most marine photosynthetic eukaryotes.

Quantitative elemental imaging in eukaryotic algae.

All organisms, fundamentally, are made from the same raw material, namely the elements of the periodic table. Biochemical diversity is achieved by how these elements are utilized, for what purpose, and in which physical location. Determining elemental distributions, especially those of trace elements that facilitate metabolism as cofactors in the active centers of essential enzymes, can determine the state of metabolism, the nutritional status, or the developmental stage of an organism. Photosynthetic eukaryotes, especially algae, are excellent subjects for quantitative analysis of elemental distribution. These microbes utilize unique metabolic pathways that require various trace nutrients at their core to enable their operation. Photosynthetic microbes also have important environmental roles as primary producers in habitats with limited nutrient supplies or toxin contaminations. Accordingly, photosynthetic eukaryotes are of great interest for biotechnological exploitation, carbon sequestration, and bioremediation, with many of the applications involving various trace elements and consequently affecting their quota and intracellular distribution. A number of diverse applications were developed for elemental imaging, allowing subcellular resolution, with X-ray fluorescence microscopy (XFM, XRF) being at the forefront, enabling quantitative descriptions of intact cells in a non-destructive method. This Tutorial Review summarizes the workflow of a quantitative, single-cell elemental distribution analysis of a eukaryotic alga using XFM.

Genomic insights into the coupling of a Chlorella-like microeukaryote and sulfur bacteria in the chemocline of permanently stratified Lake Cadagno

Meromictic Lake Cadagno is a permanently stratified system with a persistent microbial bloom within the oxic-anoxic boundary called the chemocline. The association between oxygenic and anoxygenic photosynthesis within the chemocline has been known for at least two decades. Although anoxygenic purple and green sulfur bacteria have been well studied, reports on oxygenic phytoplankton have remained sparse since their discovery in the 1920s. Nearly a century later, this study presents the first near-complete genome of a photosynthetic microbial eukaryote from the chemocline of Lake Cadagno, provisionally named Chlorella-like MAG. The 18.9 Mbp nuclear genome displays a high GC content (71.5%), and the phylogenetic placement suggests that it is a novel species of the genus Chlorella of Chlorophytes. Functional annotation of the Chlorella-like metagenome-assembled genome predicted 10,732 protein-coding genes, with an approximate 0.6% proportion potentially involved in carbon, sulfur, and nitrogen (C, N, and S) metabolism. In addition to C4 photosynthesis, this study detected genes for heat shock proteins (HSPs) in the Chlorella-like algae, consistent with the other Chlorella species. Altogether, the genomic insights in this study suggest the cooperation of photosynthetic algae with phototrophic sulfur bacteria via C, N, and S metabolism, which may aid their collective persistence in the Lake Cadagno chemocline. Furthermore, this work additionally presents the chloroplast genome of Cryptomonas-like species, which was likely to be presumed as cyanobacteria in previous studies because of the presence of phycobilisomes.

Ecological divergence of a mesocosm in an eastern boundary upwelling system assessed with multi-marker environmental DNA metabarcoding

Abstract. Eastern boundary upwelling systems (EBUS) contribute a disproportionate fraction of the global fish catch relative to their size and are especially susceptible to global environmental change. Here we present the evolution of communities over 50 d in an in situ mesocosm 6 km offshore of Callao, Peru, and in the nearby unenclosed coastal Pacific Ocean. The communities were monitored using multi-marker environmental DNA (eDNA) metabarcoding and flow cytometry. DNA extracted from weekly water samples were subjected to amplicon sequencing for four genetic loci: (1) the V1–V2 region of the 16S rRNA gene for photosynthetic eukaryotes (via their chloroplasts) and bacteria; (2) the V9 region of the 18S rRNA gene for exploration of eukaryotes but targeting phytoplankton; (3) cytochrome oxidase I (COI) for exploration of eukaryotic taxa but targeting invertebrates; and (4) the 12S rRNA gene, targeting vertebrates. The multi-marker approach showed a divergence of communities (from microbes to fish) between the mesocosm and the unenclosed ocean. Together with the environmental information, the genetic data furthered our mechanistic understanding of the processes that are shaping EBUS communities in a changing ocean. The unenclosed ocean experienced significant variability over the course of the 50 d experiment, with temporal shifts in community composition, but remained dominated by organisms that are characteristic of high-nutrient upwelling conditions (e.g., diatoms, copepods, anchovies). A large directional change was found in the mesocosm community. The mesocosm community that developed was characteristic of upwelling regions when upwelling relaxes and waters stratify (e.g., dinoflagellates, nanoflagellates). The selection of dinoflagellates under the salinity-driven experimentally stratified conditions in the mesocosm, as well as the warm conditions brought about by the coastal El Niño, may be an indication of how EBUS will respond under the global environmental changes (i.e., increases in surface temperature and freshwater input, leading to increased stratification) forecast by the IPCC.

Emerging trends in nitrogen and phosphorus signalling in photosynthetic eukaryotes.

Phosphorus (P) and nitrogen (N) are the major nutrients that constrain plant and algal growth in nature. Recent advances in understanding nutrient signalling mechanisms of these organisms have revealed molecular attributes to optimise N and P acquisition. This has illuminated the importance of interplay between N and P regulatory networks, highlighting a need to study synergistic interactions rather than single-nutrient effects. Emerging insights of nutrient signalling in polyphyletic model plants and algae hint that, although core P-starvation signalling components are conserved, distinct mechanisms for P (and N) sensing have arisen. Here, the N and P signalling mechanisms of diverse photosynthetic eukaryotes are examined, drawing parallels and differences between taxa. Future directions to understand their molecular basis, evolution, and ecology are proposed.