The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

Evolving circuitries in plant signaling cascades.

Land plants are astounding processors of information; due to their sessile nature, they adjust the molecular programs that define their development and physiology in accordance with the environment in which they dwell. Transduction of the external input to the respective internal programs hinges to a large degree on molecular signaling cascades, many of which have deep evolutionary origins in the ancestors of land plants and its closest relatives, streptophyte algae. In this Review, we discuss the evolutionary history of the defining factors of streptophyte signaling cascades, circuitries that not only operate in extant land plants and streptophyte algae, but that also likely operated in their extinct algal ancestors hundreds of millions of years ago. We hope this Review offers a starting point for future studies on the evolutionary mechanisms contributing to the current diversity and complexity of plant signaling pathways, with an emphasis on recognizing potential biases.

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.

Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

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.

Evolutionary assembly of the plant terrestrialization toolkit from protein domains.

Land plants (embryophytes) came about in a momentous evolutionary singularity: plant terrestrialization. This event marks not only the conquest of land by plants but also the massive radiation of embryophytes into a diverse array of novel forms and functions. The unique suite of traits present in the earliest land plants is thought to have been ushered in by a burst in genomic novelty. Here, we asked the question of how these bursts were possible. For this, we explored: (i) the initial emergence and (ii) the reshuffling of domains to give rise to hallmark environmental response genes of land plants. We pinpoint that a quarter of the embryophytic genes for stress physiology are specific to the lineage, yet a significant portion of this novelty arises not de novo but from reshuffling and recombining of pre-existing domains. Our data suggest that novel combinations of old genomic substrate shaped the plant terrestrialization toolkit, including hallmark processes in signalling, biotic interactions and specialized metabolism.

Chloroplast ion homeostasis - what do we know and where should we go?

Plant yields heavily depend on proper macro- and micronutrient supply from the soil. In the leaf cells, nutrient ions fulfill specific roles in biochemical reactions, especially photosynthesis housed in the chloroplast. Here, a well-balanced ion homeostasis is maintained by a number of ion transport proteins embedded in the envelope and thylakoid membranes. Ten years ago, the first alkali metal transporters from the K+ EFFLUX ANTIPORTER family were discovered in the model plant Arabidopsis. Since then, our knowledge about the physiological importance of these carriers and their substrates has greatly expanded. New insights into the role of alkali ions in plastid gene expression and photoprotective mechanisms, both prerequisites for plant productivity in natural environments, were gained. The discovery of a Cl- channel in the thylakoid and several additional plastid alkali and alkali metal transport proteins have advanced the field further. Nevertheless, scientists still have long ways to go before a complete systemic understanding of the chloroplast's ion transportome will emerge. In this Tansley review, we highlight and discuss the achievements of the last decade. More importantly, we make recommendations on what areas to prioritize, so the field can reach the next milestones. One area, laid bare by our similarity-based comparisons among phototrophs is our lack of knowledge what ion transporters are used by cyanobacteria to buffer photosynthesis fluctuations.

Constitutive activation of ABA receptors in Arabidopsis reveals unique regulatory circuitries.

Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co-receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear. We took a gain-of-function approach to study receptor-specific functionality. First, we introduced activating mutations that enforce active ABA-bound receptor conformation. We then transformed Arabidopsis ABA-deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype. Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism. We propose that ABA signaling - from perception to response - flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries.

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.

LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

Widespread occurrence of covalent lysine-cysteine redox switches in proteins.

We recently reported the discovery of a lysine-cysteine redox switch in proteins with a covalent nitrogen-oxygen-sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur-oxygen-nitrogen-oxygen-sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors, linking lysine chemistry and redox biology as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.

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). Although Embryophyta make up the major share of species numbers in Streptophyta, there is a diversity of probably >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, hence particularly on the streptophyte algae. Phylogenomic efforts have not only clarified the position of streptophyte algae relative 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 signalling 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.

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.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

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.

Green land: Multiple perspectives on green algal evolution and the earliest land plants.

Green plants, broadly defined as green algae and the land plants (together, Viridiplantae), constitute the primary eukaryotic lineage that successfully colonized Earth's emergent landscape. Members of various clades of green plants have independently made the transition from fully aquatic to subaerial habitats many times throughout Earth's history. The transition, from unicells or simple filaments to complex multicellular plant bodies with functionally differentiated tissues and organs, was accompanied by innovations built upon a genetic and phenotypic toolkit that have served aquatic green phototrophs successfully for at least a billion years. These innovations opened an enormous array of new, drier places to live on the planet and resulted in a huge diversity of land plants that have dominated terrestrial ecosystems over the past 500 million years. This review examines the greening of the land from several perspectives, from paleontology to phylogenomics, to water stress responses and the genetic toolkit shared by green algae and plants, to the genomic evolution of the sporophyte generation. We summarize advances on disparate fronts in elucidating this important event in the evolution of the biosphere and the lacunae in our understanding of it. We present the process not as a step-by-step advancement from primitive green cells to an inevitable success of embryophytes, but rather as a process of adaptations and exaptations that allowed multiple clades of green plants, with various combinations of morphological and physiological terrestrialized traits, to become diverse and successful inhabitants of the land habitats of Earth.

Tissue Resistance Decrease during Irreversible Electroporation of Pancreatic Cancer as a Biomarker for the Adaptive Immune Response and Survival.

PURPOSE: To correlate irreversible electroporation (IRE) procedural resistance changes with survival outcomes and the IRE-induced systemic immune response in patients with locally advanced pancreatic cancer (LAPC). MATERIALS AND METHODS: Data on IRE procedural tissue resistance (R) features and survival outcomes were collected from patients with LAPC treated within the context of 2 prospective clinical trials in a single tertiary center. Preprocedural and postprocedural peripheral blood samples were prospectively collected for immune monitoring. The change (ie, decrease) in R during the first 10 test pulses (ΔR10p) and during the total procedure (ΔRtotal) were calculated. Patients were divided in 2 groups on the basis of the median change in R (large ΔR vs small ΔR) and compared for differences in overall survival (OS) and progression-free survival and immune cell subsets. RESULTS: A total of 54 patients were included; of these, 20 underwent immune monitoring. Linear regression modeling showed that the first 10 test pulses reflected the change in tissue resistance during the total procedure appropriately (P < .001; R2 = 0.91). A large change in tissue resistance significantly correlated with a better OS (P = .026) and longer time to disease progression (P = .045). Furthermore, a large change in tissue resistance was associated with CD8+ T cell activation through significant upregulation of Ki-67+ (P = .02) and PD-1+ (P = .047). Additionally, this subgroup demonstrated significantly increased expression of CD80 on conventional dendritic cells (cDC1; P = .027) and PD-L1 on immunosuppressive myeloid-derived suppressor cells (P = .039). CONCLUSIONS: IRE procedural resistance changes may serve as a biomarker for survival and IRE-induced systemic CD8+ T cell and cDC1 activation.

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.

Spontaneous haemoperitoneum in pregnancy: Nationwide surveillance and Delphi audit system.

OBJECTIVE: To evaluate the incidence, diagnostic management strategies and clinical outcomes of women with spontaneous haemoperitoneum in pregnancy (SHiP) and reassess the definition of SHiP. DESIGN: A population-based cohort study using the Netherlands Obstetric Surveillance System (NethOSS). SETTING: Nationwide, the Netherlands. POPULATION: All pregnant women between April 2016 and April 2018. METHODS: This is a case study of SHiP using the monthly registry reports of NethOSS. Complete anonymised case files were obtained. A newly introduced online Delphi audit system (DAS) was used to evaluate each case, to make recommendations on improving the management of SHiP and to propose a new definition of SHiP. MAIN OUTCOME MEASURES: Incidence and outcomes, lessons learned about clinical management and the critical appraisal of the current definition of SHiP. RESULTS: In total, 24 cases were reported. After a Delphi procedure, 14 cases were classified as SHiP. The nationwide incidence was 4.9 per 100 000 births. Endometriosis and conceiving after artificial reproductive techniques were identified as risk factors. No maternal and three perinatal deaths occurred. Based on the DAS, adequate imaging of free intra-abdominal fluid, and identifying and treating women with signs of hypovolemic shock could improve the early detection and management of SHiP. A revised definition of SHiP was proposed, excluding the need for surgical or radiological intervention. CONCLUSIONS: SHiP is a rare and easily misdiagnosed condition that is associated with high perinatal mortality. To improve care, better awareness among healthcare workers is needed. The DAS is a sufficient tool to audit maternal morbidity and mortality.