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.

Repeated co-option of HMG-box genes for sex determination in brown algae and animals.

In many eukaryotes, genetic sex determination is not governed by XX/XY or ZW/ZZ systems but by a specialized region on the poorly studied U (female) or V (male) sex chromosomes. Previous studies have hinted at the existence of a dominant male-sex factor on the V chromosome in brown algae, a group of multicellular eukaryotes distantly related to animals and plants. The nature of this factor has remained elusive. Here, we demonstrate that an HMG-box gene acts as the male-determining factor in brown algae, mirroring the role HMG-box genes play in sex determination in animals. Over a billion-year evolutionary timeline, these lineages have independently co-opted the HMG box for male determination, representing a paradigm for evolution's ability to recurrently use the same genetic "toolkit" to accomplish similar tasks.

Interactions between U and V sex chromosomes during the life cycle of Ectocarpus.

In many animals and flowering plants, sex determination occurs in the diploid phase of the life cycle with XX/XY or ZW/ZZ sex chromosomes. However, in early diverging plants and most macroalgae, sex is determined by female (U) or male (V) sex chromosomes in a haploid phase called the gametophyte. Once the U and V chromosomes unite at fertilization to produce a diploid sporophyte, sex determination no longer occurs, raising key questions about the fate of the U and V sex chromosomes in the sporophyte phase. Here, we investigate genetic and molecular interactions of the UV sex chromosomes in both the haploid and diploid phases of the brown alga Ectocarpus. We reveal extensive developmental regulation of sex chromosome genes across its life cycle and implicate the TALE-HD transcription factor OUROBOROS in suppressing sex determination in the diploid phase. Small RNAs may also play a role in the repression of a female sex-linked gene, and transition to the diploid sporophyte coincides with major reconfiguration of histone H3K79me2, suggesting a more intricate role for this histone mark in Ectocarpus development than previously appreciated.

Near telomere-to-telomere genome of the model plant Physcomitrium patens.

The model plant Physcomitrium patens has played a pivotal role in enhancing our comprehension of plant evolution and development. However, the current genome harbours numerous regions that remain unfinished and erroneous. To address these issues, we generated an assembly using Oxford Nanopore reads and Hi-C mapping. The assembly incorporates telomeric and centromeric regions, thereby establishing it as a near telomere-to-telomere genome except a region in chromosome 1 that is not fully assembled due to its highly repetitive nature. This near telomere-to-telomere genome resolves the chromosome number at 26 and provides a gap-free genome assembly as well as updated gene models to aid future studies using this model organism.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Adaptive evolution of the enigmatic Takakia now facing climate change in Tibet.

The most extreme environments are the most vulnerable to transformation under a rapidly changing climate. These ecosystems harbor some of the most specialized species, which will likely suffer the highest extinction rates. We document the steepest temperature increase (2010-2021) on record at altitudes of above 4,000 m, triggering a decline of the relictual and highly adapted moss Takakia lepidozioides. Its de-novo-sequenced genome with 27,467 protein-coding genes includes distinct adaptations to abiotic stresses and comprises the largest number of fast-evolving genes under positive selection. The uplift of the study site in the last 65 million years has resulted in life-threatening UV-B radiation and drastically reduced temperatures, and we detected several of the molecular adaptations of Takakia to these environmental changes. Surprisingly, specific morphological features likely occurred earlier than 165 mya in much warmer environments. Following nearly 400 million years of evolution and resilience, this species is now facing extinction.

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.

Co-action of COP1, SPA and cryptochrome in light signal transduction and photomorphogenesis of the moss Physcomitrium patens.

The Arabidopsis COP1/SPA ubiquitin ligase suppresses photomorphogenesis in darkness. In the light, photoreceptors inactivate COP1/SPA to allow a light response. While SPA genes are specific to the green lineage, COP1 also exists in humans. This raises the question of when in evolution plant COP1 acquired the need for SPA accessory proteins. We addressed this question by generating Physcomitrium Ppcop1 mutants and comparing their visible and molecular phenotypes with those of Physcomitrium Ppspa mutants. The phenotype of Ppcop1 nonuple mutants resembles that of Ppspa mutants. Most importantly, both mutants produce green chloroplasts in complete darkness. They also exhibit dwarfed gametophores, disturbed branching of protonemata and absent gravitropism. RNA-sequencing analysis indicates that both mutants undergo weak constitutive light signaling in darkness. PpCOP1 and PpSPA proteins form a complex and they interact via their WD repeat domains with the VP motif of the cryptochrome CCE domain in a blue light-dependent manner. This resembles the interaction of Arabidopsis SPA proteins with Arabidopsis CRY1, and is different from that with Arabidopsis CRY2. Taken together, the data indicate that PpCOP1 and PpSPA act together to regulate growth and development of Physcomitrium. However, in contrast to their Arabidopsis orthologs, PpCOP1 and PpSPA proteins execute only partial suppression of light signaling in darkness. Hence, additional repressors may exist that contribute to the repression of a light response in dark-exposed Physcomitrium.

An overview of bioinformatics, genomics, and transcriptomics resources for bryophytes.

Bryophytes are useful models for the study of plant evolution, development, plant-fungal symbiosis, stress responses, and gametogenesis. Additionally, their dominant haploid gametophytic phase makes them great models for functional genomics research, allowing straightforward genome editing and gene knockout via CRISPR or homologous recombination. Until 2016, however, the only bryophyte genome sequence published was that of Physcomitrium patens. Throughout recent years, several other bryophyte genomes and transcriptome datasets became available, enabling better comparative genomics in evolutionary studies. The increase in the number of bryophyte genome and transcriptome resources available has yielded a plethora of annotations, databases, and bioinformatics tools to access the new data, which covers the large diversity of this clade and whose biology comprises features such as association with arbuscular mycorrhiza fungi, sex chromosomes, low gene redundancy, or loss of RNA editing genes for organellar transcripts. Here we provide a guide to resources available for bryophytes with regards to genome and transcriptome databases and bioinformatics tools.

A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens.

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting Physcomitrium patens that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named Physcomitrium patens amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of P. patens, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus-host coevolution.

Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis.

Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants.

KEY MESSAGE: Bryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction. With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations. Several key regulators that control the bauplan of either generation are already known. Analyses of such regulators in flowering plants are difficult due to the highly reduced gametophytic generation, and the fact that loss of function of such genes often is embryo lethal in homozygous plants. Here we set out to determine gene function and conservation via studies in bryophytes. Bryophytes are sister to vascular plants and hence allow evolutionary inferences. Moreover, embryo lethal mutants can be grown and vegetatively propagated due to the dominance of the bryophyte gametophytic generation. We determined candidates by selecting single copy orthologs that are involved in transcriptional control, and of which flowering plant mutants show defects during sexual reproduction, with a focus on the under-studied male germ line. We selected two orthologs, SWI3a/b and HAG1, and analyzed loss-of-function mutants in the moss P. patens. In both mutants, due to lack of fertile spermatozoids, fertilization and hence the switch to the diploid generation do not occur. Pphag1 additionally shows arrested male and impaired female gametangia development. We analyzed HAG1 in the dioecious liverwort M. polymorpha and found that in Mphag1 the development of gametangiophores is impaired. Taken together, we find that involvement of both regulators in sexual reproduction is conserved since the earliest divergence of land plants.

Single Nucleotide Polymorphism Charting of P. patens Reveals Accumulation of Somatic Mutations During in vitro Culture on the Scale of Natural Variation by Selfing.

Introduction: Physcomitrium patens (Hedw.) Mitten (previously known as Physcomitrella patens) was collected by H.L.K. Whitehouse in Gransden Wood (Huntingdonshire, United Kingdom) in 1962 and distributed across the globe starting in 1974. Hence, the Gransden accession has been cultured in vitro in laboratories for half a century. Today, there are more than 13 different pedigrees derived from the original accession. Additionally, accessions from other sites worldwide were collected during the last decades. Methods and Results: In this study, 250 high throughput RNA sequencing (RNA-seq) samples and 25 gDNA samples were used to detect single nucleotide polymorphisms (SNPs). Analyses were performed using five different P. patens accessions and 13 different Gransden pedigrees. SNPs were overlaid with metadata and known phenotypic variations. Unique SNPs defining Gransden pedigrees and accessions were identified and experimentally confirmed. They can be successfully employed for PCR-based identification. Conclusion: We show independent mutations in different Gransden laboratory pedigrees, demonstrating that somatic mutations occur and accumulate during in vitro culture. The frequency of such mutations is similar to those observed in naturally occurring populations. We present evidence that vegetative propagation leads to accumulation of deleterious mutations, and that sexual reproduction purges those. Unique SNP sets for five different P. patens accessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determine P. patens accessions and Gransden pedigrees are presented.

Rocket Science: The Effect of Spaceflight on Germination Physiology, Ageing, and Transcriptome of Eruca sativa Seeds.

In the 'Rocket Science' project, storage of Eruca sativa (salad rocket) seeds for six months on board the International Space Station resulted in delayed seedling establishment. Here we investigated the physiological and molecular mechanisms underpinning the spaceflight effects on dry seeds. We found that 'Space' seed germination vigor was reduced, and ageing sensitivity increased, but the spaceflight did not compromise seed viability and the development of normal seedlings. Comparative analysis of the transcriptomes (using RNAseq) in dry seeds and upon controlled artificial ageing treatment (CAAT) revealed differentially expressed genes (DEGs) associated with spaceflight and ageing. DEG categories enriched by spaceflight and CAAT included transcription and translation with reduced transcript abundances for 40S and 60S ribosomal subunit genes. Among the 'spaceflight-up' DEGs were heat shock proteins (HSPs), DNAJ-related chaperones, a heat shock factor (HSFA7a-like), and components of several DNA repair pathways (e.g., ATM, DNA ligase 1). The 'response to radiation' category was especially enriched in 'spaceflight-up' DEGs including HSPs, catalases, and the transcription factor HY5. The major finding from the physiological and transcriptome analysis is that spaceflight causes vigor loss and partial ageing during air-dry seed storage, for which space environmental factors and consequences for seed storage during spaceflights are discussed.

Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model.

Defects in flagella/cilia are often associated with infertility and disease. Motile male gametes (sperm cells) are an ancestral eukaryotic trait that has been lost in several lineages like flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) ecotypes to explore spermatozoid function. We compare genetic and epigenetic variation as well as expression profiles between the Gransden and Reute ecotype to identify a set of candidate genes associated with moss male infertility. We generated a loss-of-function mutant of a coiled-coil domain containing 39 (ccdc39) gene that is part of the flagellar hydin network. Defects in mammal and algal homologues of this gene coincide with a loss of fertility, demonstrating the evolutionary conservation of flagellar function related to male fertility across kingdoms. The Ppccdc39 mutant resembles the Gransden phenotype in terms of male fertility. Potentially, several somatic (epi-)mutations occurred during prolonged vegetative propagation of Gransden, causing regulatory differences of for example the homeodomain transcription factor BELL1. Probably these somatic changes are causative for the observed male fertility defect. We propose that moss spermatozoids might be employed as an easily accessible system to study male infertility of humans and animals in terms of flagellar structure and movement.

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens.

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo-devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web-based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de.

Genome Improvement and Genetic Map Construction for Aethionema arabicum, the First Divergent Branch in the Brassicaceae Family.

The genus Aethionema is a sister-group to the core-group of the Brassicaceae family that includes Arabidopsis thaliana and the Brassica crops. Thus, Aethionema is phylogenetically well-placed for the investigation and understanding of genome and trait evolution across the family. We aimed to improve the quality of the reference genome draft version of the annual species Aethionema arabicum Second, we constructed the first Ae. arabicum genetic map. The improved reference genome and genetic map enabled the development of each other. We started with the initially published genome (version 2.5). PacBio and MinION sequencing together with genetic map v2.5 were incorporated to produce the new reference genome v3.0. The improved genome contains 203 MB of sequence, with approximately 94% of the assembly made up of called (non-gap) bases, assembled into 2,883 scaffolds (with only 6% of the genome made up of non-called bases (Ns)). The N50 (10.3 MB) represents an 80-fold increase over the initial genome release. We generated a Recombinant Inbred Line (RIL) population that was derived from two ecotypes: Cyprus and Turkey (the reference genotype. Using a Genotyping by Sequencing (GBS) approach, we generated a high-density genetic map with 749 (v2.5) and then 632 SNPs (v3.0) was generated. The genetic map and reference genome were integrated, thus greatly improving the scaffolding of the reference genome into 11 linkage groups. We show that long-read sequencing data and genetics are complementary, resulting in an improved genome assembly in Ae. arabicum They will facilitate comparative genetic mapping work for the Brassicaceae family and are also valuable resources to investigate wide range of life history traits in Aethionema.

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.

The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data.

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.