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.

Aethionema arabicum dimorphic seed trait resetting during transition to seedlings.

The transition from germinating seeds to emerging seedlings is one of the most vulnerable plant life cycle stages. Heteromorphic diaspores (seed and fruit dispersal units) are an adaptive bet-hedging strategy to cope with spatiotemporally variable environments. While the roles and mechanisms of seedling traits have been studied in monomorphic species, which produce one type of diaspore, very little is known about seedlings in heteromorphic species. Using the dimorphic diaspore model Aethionema arabicum (Brassicaceae), we identified contrasting mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained from IND fruits by pericarp (fruit coat) removal. What follows the completion of germination is the pre-emergence seedling growth phase, which we investigated by comparative growth assays of early seedlings derived from the M+ seeds, bare M- seeds, and IND fruits. The dimorphic seedlings derived from M+ and M- seeds did not differ in their responses to ambient temperature and water potential. The phenotype of seedlings derived from IND fruits differed in that they had bent hypocotyls and their shoot and root growth was slower, but the biomechanical hypocotyl properties of 15-day-old seedlings did not differ between seedlings derived from germinated M+ seeds, M- seeds, or IND fruits. Comparison of the transcriptomes of the natural dimorphic diaspores, M+ seeds and IND fruits, identified 2,682 differentially expressed genes (DEGs) during late germination. During the subsequent 3 days of seedling pre-emergence growth, the number of DEGs was reduced 10-fold to 277 root DEGs and 16-fold to 164 shoot DEGs. Among the DEGs in early seedlings were hormonal regulators, in particular for auxin, ethylene, and gibberellins. Furthermore, DEGs were identified for water and ion transporters, nitrate transporter and assimilation enzymes, and cell wall remodeling protein genes encoding enzymes targeting xyloglucan and pectin. We conclude that the transcriptomes of seedlings derived from the dimorphic diaspores, M+ seeds and IND fruits, undergo transcriptional resetting during the post-germination pre-emergence growth transition phase from germinated diaspores to growing seedlings.

The dimorphic diaspore model Aethionema arabicum (Brassicaceae): Distinct molecular and morphological control of responses to parental and germination temperatures.

Plants in habitats with unpredictable conditions often have diversified bet-hedging strategies that ensure fitness over a wider range of variable environmental factors. A striking example is the diaspore (seed and fruit) heteromorphism that evolved to maximize species survival in Aethionema arabicum (Brassicaceae) in which external and endogenous triggers allow the production of two distinct diaspores on the same plant. Using this dimorphic diaspore model, we identified contrasting molecular, biophysical, and ecophysiological mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained by pericarp (fruit coat) removal from IND fruits. Large-scale comparative transcriptome and hormone analyses of M+ seeds, IND fruits, and M- seeds provided comprehensive datasets for their distinct thermal responses. Morph-specific differences in co-expressed gene modules in seeds, as well as in seed and pericarp hormone contents, identified a role of the IND pericarp in imposing coat dormancy by generating hypoxia affecting ABA sensitivity. This involved expression of morph-specific transcription factors, hypoxia response and cell wall-remodeling genes, as well as altered abscisic acid (ABA) metabolism, transport, and signaling. Parental temperature affected ABA contents and ABA-related gene expression and altered IND pericarp biomechanical properties. Elucidating the molecular framework underlying the diaspore heteromorphism can provide insight into developmental responses to globally changing temperatures.

Genomic imprints of unparalleled growth.

Chlorella ohadii was isolated from desert biological soil crusts, one of the harshest habitats on Earth, and is emerging as an exciting new green model for studying growth, photosynthesis and metabolism under a wide range of conditions. Here, we compared the genome of C. ohadii, the fastest growing alga on record, to that of other green algae, to reveal the genomic imprints empowering its unparalleled growth rate and resistance to various stressors, including extreme illumination. This included the genome of its close relative, but slower growing and photodamage sensitive, C. sorokiniana UTEX 1663. A larger number of ribosome-encoding genes, high intron abundance, increased codon bias and unique genes potentially involved in metabolic flexibility and resistance to photodamage are all consistent with the faster growth of C. ohadii. Some of these characteristics highlight general trends in Chlorophyta and Chlorella spp. evolution, and others open new broad avenues for mechanistic exploration of their relationship with growth. This work entails a unique case study for the genomic adaptations and costs of exceptionally fast growth and sheds light on the genomic signatures of fast growth in photosynthetic cells. It also provides an important resource for future studies leveraging the unique properties of C. ohadii for photosynthesis and stress response research alongside their utilization for synthetic biology and biotechnology aims.

Enhanced Susceptibility to Tomato Chlorosis Virus (ToCV) in Hsp90- and Sgt1-Silenced Plants: Insights from Gene Expression Dynamics.

The emerging whitefly-transmitted crinivirus tomato chlorosis virus (ToCV) causes substantial economic losses by inducing yellow leaf disorder in tomato crops. This study explores potential resistance mechanisms by examining early-stage molecular responses to ToCV. A time-course transcriptome analysis compared naïve, mock, and ToCV-infected plants at 2, 7, and 14 days post-infection (dpi). Gene expression changes were most notable at 2 and 14 dpi, likely corresponding to whitefly feeding and viral infection. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed key genes and pathways associated with ToCV infection, including those related to plant immunity, flavonoid and steroid biosynthesis, photosynthesis, and hormone signaling. Additionally, virus-derived small interfering RNAs (vsRNAs) originating from ToCV predominantly came from RNA2 and were 22 nucleotides in length. Furthermore, two genes involved in plant immunity, Hsp90 (heat shock protein 90) and its co-chaperone Sgt1 (suppressor of the G2 allele of Skp1) were targeted through viral-induced gene silencing (VIGS), showing a potential contribution to basal resistance against viral infections since their reduction correlated with increased ToCV accumulation. This study provides insights into tomato plant responses to ToCV, with potential implications for developing effective disease control strategies.

Transcriptomic Insight into the Pollen Tube Growth of Olea europaea L. subsp. europaea Reveals Reprogramming and Pollen-Specific Genes Including New Transcription Factors.

The pollen tube is a key innovation of land plants that is essential for successful fertilisation. Its development and growth have been profusely studied in model organisms, but in spite of the economic impact of olive trees, little is known regarding the genome-wide events underlying pollen hydration and growth in this species. To fill this gap, triplicate mRNA samples at 0, 1, 3, and 6 h of in vitro germination of olive cultivar Picual pollen were analysed by RNA-seq. A bioinformatics R workflow called RSeqFlow was developed contemplating the best practices described in the literature, covering from expression data filtering to differential expression and clustering, to finally propose hub genes. The resulting olive pollen transcriptome consisted of 22,418 reliable transcripts, where 5364 were differentially expressed, out of which 173 have no orthologue in plants and up to 3 of them might be pollen-specific transcription factors. Functional enrichment revealed a deep transcriptional reprogramming in mature olive pollen that is also dependent on protein stability and turnover to allow pollen tube emergence, with many hub genes related to heat shock proteins and F-box-containing proteins. Reprogramming extends to the first 3 h of growth, including processes consistent with studies performed in other plant species, such as global down-regulation of biosynthetic processes, vesicle/organelle trafficking and cytoskeleton remodelling. In the last stages, growth should be maintained from persistent transcripts. Mature pollen is equipped with transcripts to successfully cope with adverse environments, even though the in vitro growth seems to induce several stress responses. Finally, pollen-specific transcription factors were proposed as probable drivers of pollen germination in olive trees, which also shows an overall increased number of pollen-specific gene isoforms relative to other plants.

PEATmoss: A Gene Expression Atlas for Bryophytes.

PEATmoss is an interactive gene expression atlas for bryophytes, which originally unified Physcomitrium patens RNA-seq and microarray expression data from multiple gene annotation versions. This atlas includes more than 100 experiments of P. patens, is expanding to host Anthoceros agrestis and Marchantia polymorpha, and aims to host data from more species in the future. PEATmoss has multiple visualization methods and tools for data downloading and is connected to the Physcomitrium patens Gene Model Lookup DB (PpGML DB), which links P. patens genes to annotations and resources from several databases and contains tools for gene version lookup and sequence and annotation extraction. Among the new features available in PEATmoss are dataset privacy control, multispecies menu, interactive color scale, co-expression network visualization, and replicate data downloading.

MangoBase: A Genomics Portal and Gene Expression Atlas for Mangifera indica.

Mango (Mangifera indica L.) (2n = 40) is a member of the Anacardiaceae family, which was domesticated at least 4000 years ago in Asia. Mangoes are delicious fruits with great nutritional value. They are one of the major fruit crops worldwide, cultivated in more than 100 countries, with a production of more than 40 million tons. Recently the genome sequences of several mango varieties have been released, but there are no bioinformatics platforms dedicated to mango genomics and breeding to host mango omics data. Here, we present MangoBase, a web portal dedicated to mango genomics, which provides multiple interactive bioinformatics tools, sequences, and annotations to analyze, visualize, and download omics data related to mango. Additionally, MangoBase includes a gene expression atlas with 12 datasets and 80 experiments representing some of the most significant mango RNA-seq experiments published to this date. These experiments study mango fruit ripening in several cultivars with different pulp firmness and sweetness or peel coloration, and other experiments also study hot water postharvest treatment, infection with C. gloeosporioides, and the main mango tree organ tissues.

OliveAtlas: A Gene Expression Atlas Tool for Olea europaea.

The olive (Olea europaea L.) is an ancient crop of great importance in the Mediterranean basin due to the production of olive oil and table olives, which are important sources of fat and have benefits for human health. This crop is expanding and increasing its production worldwide and five olive genomes have recently been sequenced, representing a wild olive and important cultivars in terms of olive oil production, intensive agriculture, and adaptation to the East Asian climate. However, few bioinformatic and genomic resources are available to assist olive research and breeding, and there are no platforms to query olive gene expression data. Here, we present OliveAtlas, an interactive gene expression atlas for olive with multiple bioinformatics tools and visualization methods, enabling multiple gene comparison, replicate inspection, gene set enrichment, and data downloading. It contains 70 RNA-seq experiments, organized in 10 data sets representing the main olive plant organs, the pollen germination and pollen tube elongation process, and the response to a collection of biotic and abiotic stresses, among other experimental conditions. OliveAtlas is a web tool based on easyGDB with expression data based on the 'Picual' genome reference and gene annotation.

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.

Comparative transcriptomics identifies candidate genes involved in the evolutionary transition from dehiscent to indehiscent fruits in Lepidium (Brassicaceae).

BACKGROUND: Fruits are the seed-bearing structures of flowering plants and are highly diverse in terms of morphology, texture and maturation. Dehiscent fruits split open upon maturation to discharge their seeds while indehiscent fruits are dispersed as a whole. Indehiscent fruits evolved from dehiscent fruits several times independently in the crucifer family (Brassicaceae). The fruits of Lepidium appelianum, for example, are indehiscent while the fruits of the closely related L. campestre are dehiscent. Here, we investigate the molecular and genetic mechanisms underlying the evolutionary transition from dehiscent to indehiscent fruits using these two Lepidium species as model system. RESULTS: We have sequenced the transcriptomes and small RNAs of floral buds, flowers and fruits of L. appelianum and L. campestre and analyzed differentially expressed genes (DEGs) and differently differentially expressed genes (DDEGs). DEGs are genes that show significantly different transcript levels in the same structures (buds, flowers and fruits) in different species, or in different structures in the same species. DDEGs are genes for which the change in expression level between two structures is significantly different in one species than in the other. Comparing the two species, the highest number of DEGs was found in flowers, followed by fruits and floral buds while the highest number of DDEGs was found in fruits versus flowers followed by flowers versus floral buds. Several gene ontology terms related to cell wall synthesis and degradation were overrepresented in different sets of DEGs highlighting the importance of these processes for fruit opening. Furthermore, the fruit valve identity genes FRUITFULL and YABBY3 were among the DEGs identified. Finally, the microRNA miR166 as well as the TCP transcription factors BRANCHED1 (BRC1) and TCP FAMILY TRANSCRIPTION FACTOR 4 (TCP4) were found to be DDEGs. CONCLUSIONS: Our study reveals differences in gene expression between dehiscent and indehiscent fruits and uncovers miR166, BRC1 and TCP4 as candidate genes for the evolutionary transition from dehiscent to indehiscent fruits in Lepidium.

EasyGDB: a low-maintenance and highly customizable system to develop genomics portals.

SUMMARY: EasyGDB is an easy-to-implement low-maintenance tool developed to create genomic data management web platforms. It can be used for any species, group of species, or multiple genome or annotation versions. EasyGDB provides a framework to develop a web portal that includes the general information about species, projects and members, and bioinformatics tools such as file downloads, BLAST, genome browser, annotation search, gene expression visualization, annotation and sequence download, and gene ids and orthologs lookup. The code of EasyGDB facilitates data maintenance and update for non-experienced bioinformaticians, using BLAST databases to store and retrieve sequence data in gene annotation pages and bioinformatics tools, and JSON files to customize metadata. EasyGDB is a highly customizable tool. Any section and tool can be enabled or disabled like a switch through a single configuration file. This tool aims to simplify the development of genomics portals in non-model species, providing a modern web style with embedded interactive bioinformatics tools to cover all the common needs derived from genomics projects. AVAILABILITY AND IMPLEMENTATION: The code and manual to use EasyGDB can be found at https://github.com/noefp/easy_gdb.

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.

The EpiDiverse Plant Epigenome-Wide Association Studies (EWAS) Pipeline.

Bisulfite sequencing is a widely used technique for determining DNA methylation and its relationship with epigenetics, genetics, and environmental parameters. Various techniques were implemented for epigenome-wide association studies (EWAS) to reveal meaningful associations; however, there are only very few plant studies available to date. Here, we developed the EpiDiverse EWAS pipeline and tested it using two plant datasets, from P. abies (Norway spruce) and Q. lobata (valley oak). Hence, we present an EWAS implementation tested for non-model plant species and describe its use.

EpiDiverse Toolkit: a pipeline suite for the analysis of bisulfite sequencing data in ecological plant epigenetics.

The expanding scope and scale of next generation sequencing experiments in ecological plant epigenetics brings new challenges for computational analysis. Existing tools built for model data may not address the needs of users looking to apply these techniques to non-model species, particularly on a population or community level. Here we present a toolkit suitable for plant ecologists working with whole genome bisulfite sequencing; it includes pipelines for mapping, the calling of methylation values and differential methylation between groups, epigenome-wide association studies, and a novel implementation for both variant calling and discriminating between genetic and epigenetic variation.

Gene-rich UV sex chromosomes harbor conserved regulators of sexual development.

Nonrecombining sex chromosomes, like the mammalian Y, often lose genes and accumulate transposable elements, a process termed degeneration. The correlation between suppressed recombination and degeneration is clear in animal XY systems, but the absence of recombination is confounded with other asymmetries between the X and Y. In contrast, UV sex chromosomes, like those found in bryophytes, experience symmetrical population genetic conditions. Here, we generate nearly gapless female and male chromosome-scale reference genomes of the moss Ceratodon purpureus to test for degeneration in the bryophyte UV sex chromosomes. We show that the moss sex chromosomes evolved over 300 million years ago and expanded via two chromosomal fusions. Although the sex chromosomes exhibit weaker purifying selection than autosomes, we find that suppressed recombination alone is insufficient to drive degeneration. Instead, the U and V sex chromosomes harbor thousands of broadly expressed genes, including numerous key regulators of sexual development across land plants.

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.

Aethionema arabicum genome annotation using PacBio full-length transcripts provides a valuable resource for seed dormancy and Brassicaceae evolution research.

Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed (development, regulation, germination, dormancy) and fruit (development, dehiscence mechanisms) characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae. arabicum gene annotation using 294 RNA-seq libraries and 136 307 full-length PacBio Iso-seq transcripts, increasing BUSCO completeness by 11.6% and featuring 5606 additional genes. Analysis of orthologs showed a lower number of genes in Ae. arabicum than in other Brassicaceae, which could be partially explained by loss of homeologs derived from the At-α polyploidization event and by a lower occurrence of tandem duplications after divergence of Aethionema from the other Brassicaceae. Benchmarking of MADS-box genes identified orthologs of FUL and AGL79 not found in previous versions. Analysis of full-length transcripts related to ABA-mediated seed dormancy discovered a conserved isoform of PIF6-ß and antisense transcripts in ABI3, ABI4 and DOG1, among other cases found of different alternative splicing between Turkey and Cyprus ecotypes. The presented data allow alternative splicing mining and proposition of numerous hypotheses to research evolution and functional genomics. Annotation data and sequences are available at the Ae. arabicum DB (https://plantcode.online.uni-marburg.de/aetar_db).

Transposon activation is a major driver in the genome evolution of cultivated olive trees (Olea europaea L.).

The primary domestication of olive (Olea europaea L.) in the Levant dates back to the Neolithic period, around 6,000-5,500 BC, as some archeological remains attest. Cultivated olive trees are reproduced clonally, with sexual crosses being the sporadic events that drive the development of new varieties. In order to determine the genomic changes which have occurred in a modern olive cultivar, the genome of the Picual cultivar, one of the most popular olive varieties, was sequenced. Additional 40 cultivated and 10 wild accessions were re-sequenced to elucidate the evolution of the olive genome during the domestication process. It was found that the genome of the 'Picual' cultivar contains 79,667 gene models, of which 78,079 were protein-coding genes and 1,588 were tRNA. Population analyses support two independent events in olive domestication, including an early possible genetic bottleneck. Despite genetic bottlenecks, cultivated accessions showed a high genetic diversity driven by the activation of transposable elements (TE). A high TE gene expression was observed in presently cultivated olives, which suggests a current activity of TEs in domesticated olives. Several TEs families were expanded in the last 5,000 or 6,000 years and produced insertions near genes that may have been involved in selected traits during domestication as reproduction, photosynthesis, seed development, and oil production. Therefore, a great genetic variability has been found in cultivated olive as a result of a significant activation of TEs during the domestication process.