Phylogeny and evolution of streptophyte algae.

The Streptophyta emerged about a billion years ago. Nowadays, this branch of the green lineage is most famous for one of its clades, the land plants (Embryophyta). While Embryophyta make up the major share of species numbers in Streptophyta, there is a diversity of likely more than 5000 species of streptophyte algae that form a paraphyletic grade next to land plants. Here, we focus on the deep divergences that gave rise to the diversity of streptophytes-and thus, particularly on the streptophyte algae. Phylogenomic efforts have not only clarified the position of streptophyte algae to land plants but recent efforts have also begun to unravel the relationships and major radiations within streptophyte algal diversity. We illustrate how new phylogenomic perspectives have changed our view on the evolutionary emergence of key traits such as intricate signaling networks that are intertwined with multicellular growth and the chemodiverse hotbed from which they emerged. These traits are key for the biology of land plants-but were bequeathed from their algal progenitors.

Genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

Zygnematophyceae are the algal sisters of land plants. Here we sequenced four genomes of filamentous Zygnematophyceae, including chromosome-scale assemblies for three strains of Zygnema circumcarinatum. We inferred traits in the ancestor of Zygnematophyceae and land plants that might have ushered in the conquest of land by plants: expanded genes for signaling cascades, environmental response, and multicellular growth. Zygnematophyceae and land plants share all the major enzymes for cell wall synthesis and remodifications, and gene gains shaped this toolkit. Co-expression network analyses uncover gene cohorts that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

Sequence similarity networks bear out hierarchical relationships of green cytochrome P450.

Land plants have diversified enzyme families. One of the most prominent is the cytochrome P450 (CYP or CYP450) family. With over 443,000 CYP proteins sequenced across the tree of life, CYPs are ubiquitous in archaea, bacteria, and eukaryotes. Here, we focused on land plants and algae to study the role of CYP diversification. CYPs, acting as monooxygenases, catalyze hydroxylation reactions crucial for specialized plant metabolic pathways, including detoxification and phytohormone production; the CYPome consists of one enormous superfamily that is divided into clans and families. Their evolutionary history speaks of high substrate promiscuity; radiation and functional diversification have yielded numerous CYP families. To understand the evolutionary relationships within the CYPs, we employed sequence similarity network analyses. We recovered distinct clusters representing different CYP families, reflecting their diversified sequences that we link to the prediction of functionalities. Hierarchical clustering and phylogenetic analysis further elucidated relationships between CYP clans, uncovering their shared deep evolutionary history. We explored the distribution and diversification of CYP subfamilies across plant and algal lineages, uncovering novel candidates and providing insights into the evolution of these enzyme families. This identified unexpected relationships between CYP families, such as the link between CYP82 and CYP74, shedding light on their roles in plant defense signaling pathways. Our approach provides a methodology that brings insights into the emergence of new functions within the CYP450 family, contributing to the evolutionary history of plants and algae. These insights can be further validated and implemented via experimental setups under various external conditions.

Divergent evolution of the alcohol-forming pathway of wax biosynthesis among bryophytes.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

Phylogenomic insights into the first multicellular streptophyte.

Streptophytes are best known as the clade containing the teeming diversity of embryophytes (land plants).1,2,3,4 Next to embryophytes are however a range of freshwater and terrestrial algae that bear important information on the emergence of key traits of land plants. Among these, the Klebsormidiophyceae stand out. Thriving in diverse environments-from mundane (ubiquitous occurrence on tree barks and rocks) to extreme (from the Atacama Desert to the Antarctic)-Klebsormidiophyceae can exhibit filamentous body plans and display remarkable resilience as colonizers of terrestrial habitats.5,6 Currently, the lack of a robust phylogenetic framework for the Klebsormidiophyceae hampers our understanding of the evolutionary history of these key traits. Here, we conducted a phylogenomic analysis utilizing advanced models that can counteract systematic biases. We sequenced 24 new transcriptomes of Klebsormidiophyceae and combined them with 14 previously published genomic and transcriptomic datasets. Using an analysis built on 845 loci and sophisticated mixture models, we establish a phylogenomic framework, dividing the six distinct genera of Klebsormidiophyceae in a novel three-order system, with a deep divergence more than 830 million years ago. Our reconstructions of ancestral states suggest (1) an evolutionary history of multiple transitions between terrestrial-aquatic habitats, with stem Klebsormidiales having conquered land earlier than embryophytes, and (2) that the body plan of the last common ancestor of Klebsormidiophyceae was multicellular, with a high probability that it was filamentous whereas the sarcinoids and unicells in Klebsormidiophyceae are likely derived states. We provide evidence that the first multicellular streptophytes likely lived about a billion years ago.

Divergent responses in desiccation experiments in two ecophysiologically different Zygnematophyceae.

Water scarcity can be considered a major stressor on land, with desiccation being its most extreme form. Land plants have found two different solutions to this challenge: avoidance and tolerance. The closest algal relatives to land plants, the Zygnematophyceae, use the latter, and how this is realized is of great interest for our understanding of the conquest of land. Here, we worked with two representatives of the Zygnematophyceae, Zygnema circumcarinatum SAG 698-1b and Mesotaenium endlicherianum SAG 12.97, who differ in habitats and drought resilience. We challenged both algal species with severe desiccation in a laboratory setup until photosynthesis ceased, followed by a recovery period. We assessed their morphological, photophysiological, and transcriptomic responses. Our data pinpoint global differential gene expression patterns that speak of conserved responses, from calcium-mediated signaling to the adjustment of plastid biology, cell envelopes, and amino acid pathways, between Zygnematophyceae and land plants despite their strong ecophysiological divergence. The main difference between the two species appears to rest in a readjustment of the photobiology of Zygnema, while Mesotaenium experiences stress beyond a tipping point.

Environmental gradients reveal stress hubs pre-dating plant terrestrialization.

Plant terrestrialization brought forth the land plants (embryophytes). Embryophytes account for most of the biomass on land and evolved from streptophyte algae in a singular event. Recent advances have unravelled the first full genomes of the closest algal relatives of land plants; among the first such species was Mesotaenium endlicherianum. Here we used fine-combed RNA sequencing in tandem with a photophysiological assessment on Mesotaenium exposed to a continuous range of temperature and light cues. Our data establish a grid of 42 different conditions, resulting in 128 transcriptomes and ~1.5 Tbp (~9.9 billion reads) of data to study the combinatory effects of stress response using clustering along gradients. Mesotaenium shares with land plants major hubs in genetic networks underpinning stress response and acclimation. Our data suggest that lipid droplet formation and plastid and cell wall-derived signals have denominated molecular programmes since more than 600 million years of streptophyte evolution-before plants made their first steps on land.

An ancient route towards salicylic acid and its implications for the perpetual Trichormus-Azolla symbiosis.

Despite its small size, the water fern Azolla is a giant among plant symbioses. Within each of its leaflets, a specialized leaf cavity is home to a population of nitrogen-fixing cyanobacteria (cyanobionts). Although a number of plant-cyanobiont symbioses exist, Azolla is unique in that its symbiosis is perpetual: the cyanobionts are inherited during sexual and vegetative propagation. What underpins the communication between the two partners? In angiosperms, the phytohormone salicylic acid (SA) is a well-known regulator of plant-microbe interactions. Using high-performance liquid chromatography-tandem mass spectrometry, we pinpoint the presence of SA in the fern. Comparative genomics and phylogenetics on SA biosynthesis genes across Chloroplastida reveal that the entire Phenylalanine ammonia-lyase-dependent pathway likely existed in the last common ancestor of land plants. Indeed, Azolla filiculoides secondarily lost its isochorismate synthase but has the genetic competence to derive SA from benzoic acid; the presence of SA in artificially cyanobiont-free Azolla supports the existence of this route. Global gene expression data and SA levels from cyanobiont-containing and -free A. filiculoides link SA synthesis with the symbioses: SA appears to induce cyanobacterial proliferation, whereas removal of the symbiont results in reduced SA levels in a nitrogen-dependent manner.

Fern cell walls and the evolution of arabinogalactan proteins in streptophytes.

Significant changes have occurred in plant cell wall composition during evolution and diversification of tracheophytes. As the sister lineage to seed plants, knowledge on the cell wall of ferns is key to track evolutionary changes across tracheophytes and to understand seed plant-specific evolutionary innovations. Fern cell wall composition is not fully understood, including limited knowledge of glycoproteins such as the fern arabinogalactan proteins (AGPs). Here, we characterize the AGPs from the leptosporangiate fern genera Azolla, Salvinia, and Ceratopteris. The carbohydrate moiety of seed plant AGPs consists of a galactan backbone including mainly 1,3- and 1,3,6-linked pyranosidic galactose, which is conserved across the investigated fern AGPs. Yet, unlike AGPs of angiosperms, those of ferns contained the unusual sugar 3-O-methylrhamnose. Besides terminal furanosidic arabinose, Ara (Araf), the main linkage type of Araf in the ferns was 1,2-linked Araf, whereas in seed plants 1,5-linked Araf is often dominating. Antibodies directed against carbohydrate epitopes of AGPs supported the structural differences between AGPs of ferns and seed plants. Comparison of AGP linkage types across the streptophyte lineage showed that angiosperms have rather conserved monosaccharide linkage types; by contrast bryophytes, ferns, and gymnosperms showed more variability. Phylogenetic analyses of glycosyltransferases involved in AGP biosynthesis and bioinformatic search for AGP protein backbones revealed a versatile genetic toolkit for AGP complexity in ferns. Our data reveal important differences across AGP diversity of which the functional significance is unknown. This diversity sheds light on the evolution of the hallmark feature of tracheophytes: their elaborate cell walls.

Chromosome-level genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

The filamentous and unicellular algae of the class Zygnematophyceae are the closest algal relatives of land plants. Inferring the properties of the last common ancestor shared by these algae and land plants allows us to identify decisive traits that enabled the conquest of land by plants. We sequenced four genomes of filamentous Zygnematophyceae (three strains of Zygnema circumcarinatum and one strain of Z. cylindricum) and generated chromosome-scale assemblies for all strains of the emerging model system Z. circumcarinatum. Comparative genomic analyses reveal expanded genes for signaling cascades, environmental response, and intracellular trafficking that we associate with multicellularity. Gene family analyses suggest that Zygnematophyceae share all the major enzymes with land plants for cell wall polysaccharide synthesis, degradation, and modifications; most of the enzymes for cell wall innovations, especially for polysaccharide backbone synthesis, were gained more than 700 million years ago. In Zygnematophyceae, these enzyme families expanded, forming co-expressed modules. Transcriptomic profiling of over 19 growth conditions combined with co-expression network analyses uncover cohorts of genes that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

Crossroads in the evolution of plant specialized metabolism.

The monophyletic group of embryophytes (land plants) stands out among photosynthetic eukaryotes: they are the sole constituents of the macroscopic flora on land. In their entirety, embryophytes account for the majority of the biomass on land and constitute an astounding biodiversity. What allowed for the massive radiation of this particular lineage? One of the defining features of all land plants is the production of an array of specialized metabolites. The compounds that the specialized metabolic pathways of embryophytes produce have diverse functions, ranging from superabundant structural polymers and compounds that ward off abiotic and biotic challenges, to signaling molecules whose abundance is measured at the nanomolar scale. These specialized metabolites govern the growth, development, and physiology of land plants-including their response to the environment. Hence, specialized metabolites define the biology of land plants as we know it. And they were likely a foundation for their success. It is thus intriguing to find that the closest algal relatives of land plants, freshwater organisms from the grade of streptophyte algae, possess homologs for key enzymes of specialized metabolic pathways known from land plants. Indeed, some studies suggest that signature metabolites emerging from these pathways can be found in streptophyte algae. Here we synthesize the current understanding of which routes of the specialized metabolism of embryophytes can be traced to a time before plants had conquered land.

[Hypothermia in elderly patients].

Hypothermia in older patients is a serious symptom, with high morbidity and mortality, and often an atypical presentation of an underlying problem. The most common causes are an infection and exposure to extreme cold ('accidental hypothermia'), but there are other, less common causes. These two cases show hypothermia as one of the symptoms in atypical presentations of underlying conditions. It is important to run diagnostics for infectious diseases and other underlying causes, and start antibiotics promptly. If there is no response to antibiotics and diagnostics do not reveal evidence of an infection, clinicians need to consider other causes of hypothermia.

A phylogenomically informed five-order system for the closest relatives of land plants.

The evolution of streptophytes had a profound impact on life on Earth. They brought forth those photosynthetic eukaryotes that today dominate the macroscopic flora: the land plants (Embryophyta).1 There is convincing evidence that the unicellular/filamentous Zygnematophyceae-and not the morphologically more elaborate Coleochaetophyceae or Charophyceae-are the closest algal relatives of land plants.2-6 Despite the species richness (>4,000), wide distribution, and key evolutionary position of the zygnematophytes, their internal phylogeny remains largely unresolved.7,8 There are also putative zygnematophytes with interesting body plan modifications (e.g., filamentous growth) whose phylogenetic affiliations remain unknown. Here, we studied a filamentous green alga (strain MZCH580) from an Austrian peat bog with central or parietal chloroplasts that lack discernible pyrenoids. It represents Mougeotiopsis calospora PALLA, an enigmatic alga that was described more than 120 years ago9 but never subjected to molecular analyses. We generated transcriptomic data of M. calospora strain MZCH580 and conducted comprehensive phylogenomic analyses (326 nuclear loci) for 46 taxonomically diverse zygnematophytes. Strain MZCH580 falls in a deep-branching zygnematophycean clade together with some unicellular species and thus represents a formerly unknown zygnematophycean lineage with filamentous growth. Our well-supported phylogenomic tree lets us propose a new five-order system for the Zygnematophyceae and provides evidence for at least five independent origins of true filamentous growth in the closest algal relatives of land plants. This phylogeny provides a robust and comprehensive framework for performing comparative analyses and inferring the evolution of cellular traits and body plans in the closest relatives of land plants.

Evolutionary genomic insights into cyanobacterial symbioses in plants.

Photosynthesis, the ability to fix atmospheric carbon dioxide, was acquired by eukaryotes through symbiosis: the plastids of plants and algae resulted from a cyanobacterial symbiosis that commenced more than 1.5 billion years ago and has chartered a unique evolutionary path. This resulted in the evolutionary origin of plants and algae. Some extant land plants have recruited additional biochemical aid from symbiotic cyanobacteria; these plants associate with filamentous cyanobacteria that fix atmospheric nitrogen. Examples of such interactions can be found in select species from across all major lineages of land plants. The recent rise in genomic and transcriptomic data has provided new insights into the molecular foundation of these interactions. Furthermore, the hornwort Anthoceros has emerged as a model system for the molecular biology of cyanobacteria-plant interactions. Here, we review these developments driven by high-throughput data and pinpoint their power to yield general patterns across these diverse symbioses.

Submergence of the filamentous Zygnematophyceae Mougeotia induces differential gene expression patterns associated with core metabolism and photosynthesis.

The streptophyte algal class Zygnematophyceae is the closest algal sister lineage to land plants. In nature, Zygnematophyceae can grow in both terrestrial and freshwater habitats and how they do this is an important unanswered question. Here, we studied what happens to the zygnematophyceaen alga Mougeotia sp., which usually occurs in permanent and temporary freshwater bodies, when it is shifted to liquid growth conditions after growth on a solid substrate. Using global differential gene expression profiling, we identified changes in the core metabolism of the organism interlinked with photosynthesis; the latter went hand in hand with measurable impact on the photophysiology as assessed via pulse amplitude modulation (PAM) fluorometry. Our data reveal a pronounced change in the overall physiology of the alga after submergence and pinpoint candidate genes that play a role. These results provide insight into the importance of photophysiological readjustment when filamentous Zygnematophyceae transition between terrestrial and aquatic habitats.

Underwater CAM photosynthesis elucidated by Isoetes genome.

To conserve water in arid environments, numerous plant lineages have independently evolved Crassulacean Acid Metabolism (CAM). Interestingly, Isoetes, an aquatic lycophyte, can also perform CAM as an adaptation to low CO2 availability underwater. However, little is known about the evolution of CAM in aquatic plants and the lack of genomic data has hindered comparison between aquatic and terrestrial CAM. Here, we investigate underwater CAM in Isoetes taiwanensis by generating a high-quality genome assembly and RNA-seq time course. Despite broad similarities between CAM in Isoetes and terrestrial angiosperms, we identify several key differences. Notably, Isoetes may have recruited the lesser-known 'bacterial-type' PEPC, along with the 'plant-type' exclusively used in other CAM and C4 plants for carboxylation of PEP. Furthermore, we find that circadian control of key CAM pathway genes has diverged considerably in Isoetes relative to flowering plants. This suggests the existence of more evolutionary paths to CAM than previously recognized.

Characterization of T cell responses to co-administered hookworm vaccine candidates Na-GST-1 and Na-APR-1 in healthy adults in Gabon.

Two hookworm vaccine candidates, Na-GST-1 and Na-APR-1, formulated with Glucopyranosyl Lipid A (GLA-AF) adjuvant, have been shown to be safe, well tolerated, and to induce antibody responses in a Phase 1 clinical trial (Clinicaltrials.gov NCT02126462) conducted in Gabon. Here, we characterized T cell responses in 24 Gabonese volunteers randomized to get vaccinated three times with Na-GST-1 and Na-APR-1 at doses of 30µg (n = 8) or 100µg (n = 10) and as control Hepatitis B (n = 6). Blood was collected pre- and post-vaccination on days 0, 28, and 180 as well as 2-weeks after each vaccine dose on days 14, 42, and 194 for PBMCs isolation. PBMCs were stimulated with recombinant Na-GST-1 or Na-APR-1, before (days 0, 28 and 180) and two weeks after (days 14, 42 and 194) each vaccination and used to characterize T cell responses by flow and mass cytometry. A significant increase in Na-GST-1 -specific CD4+ T cells producing IL-2 and TNF, correlated with specific IgG antibody levels, after the third vaccination (day 194) was observed. In contrast, no increase in Na-APR-1 specific T cell responses were induced by the vaccine. Mass cytometry revealed that, Na-GST-1 cytokine producing CD4+ T cells were CD161+ memory cells expressing CTLA-4 and CD40-L. Blocking CTLA-4 enhanced the cytokine response to Na-GST-1. In Gabonese volunteers, hookworm vaccine candidate, Na-GST-1, induces detectable CD4+ T cell responses that correlate with specific antibody levels. As these CD4+ T cells express CTLA-4, and blocking this inhibitory molecules resulted in enhanced cytokine production, the question arises whether this pathway can be targeted to enhance vaccine immunogenicity.