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.

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 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 abscisic acid (ABA) sensitivity. This involved expression of morph-specific transcription factors, hypoxia response, and cell wall remodeling genes, as well as altered 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.

A tale of two morphs: developmental patterns and mechanisms of seed coat differentiation in the dimorphic diaspore model Aethionema arabicum (Brassicaceae).

The developmental transition from a fertilized ovule to a dispersed diaspore (seed or fruit) involves complex differentiation processes of the ovule's integuments leading to the diversity in mature seed coat structures in angiosperms. In this study, comparative imaging and transcriptome analysis were combined to investigate the morph-specific developmental differences during outer seed coat differentiation and mucilage production in Aethionema arabicum, the Brassicaceae model for diaspore dimorphism. One of the intriguing adaptations of this species is the production and dispersal of morphologically distinct, mucilaginous and non-mucilaginous diaspores from the same plant (dimorphism). The dehiscent fruit morph programme producing multiple mucilaginous seed diaspores was used as the default trait combination, similar to Arabidopsis thaliana, and was compared with the indehiscent fruit morph programme leading to non-mucilaginous diaspores. Synchrotron-based radiation X-ray tomographic microscopy revealed a co-ordinated framework of morph-specific early changes in internal anatomy of developing A. arabicum gynoecia including seed abortion in the indehiscent programme and mucilage production by the mucilaginous seed coat. The associated comparative analysis of the gene expression patterns revealed that the unique seed coat dimorphism of Ae. arabicum provides an excellent model system for comparative study of the control of epidermal cell differentiation and mucilage biosynthesis by the mucilage transcription factor cascade and their downstream cell wall and mucilage remodelling genes. Elucidating the underlying molecular framework of the dimorphic diaspore syndrome is key to understanding differential regulation of bet-hedging survival strategies in challenging environments, timely in the face of global climatic change.

When the BRANCHED network bears fruit: how carpic dominance causes fruit dimorphism in Aethionema.

Life in unpredictably changing habitats is a great challenge, especially for sessile organisms like plants. Fruit and seed heteromorphism is one way to cope with such variable environmental conditions. It denotes the production of distinct types of fruits and seeds that often mediate distinct life-history strategies in terms of dispersal, germination and seedling establishment. But although the phenomenon can be found in numerous species and apparently evolved several times independently, its developmental time course or molecular regulation remains largely unknown. Here, we studied fruit development in Aethionema arabicum, a dimorphic member of the Brassicaceae family. We characterized fruit morph differentiation by comparatively analyzing discriminating characters like fruit growth, seed abortion and dehiscence zone development. Our data demonstrate that fruit morph determination is a 'last-minute' decision happening in flowers after anthesis directly before the first morphotypical differences start to occur. Several growth experiments in combination with hormone and gene expression analyses further indicate that an accumulation balance of the plant hormones auxin and cytokinin in open flowers together with the transcript abundance of the Ae. arabicum ortholog of BRANCHED1, encoding a transcription factor known for its conserved function as a branching repressor, may guide fruit morph determination. Thus, we hypothesize that the plasticity of the fruit morph ratio in Ae. arabicum may have evolved through the modification of a preexisting network known to govern correlative dominance between shoot organs.

Usability of reference-free transcriptome assemblies for detection of differential expression: a case study on Aethionema arabicum dimorphic seeds.

BACKGROUND: RNA-sequencing analysis is increasingly utilized to study gene expression in non-model organisms without sequenced genomes. Aethionema arabicum (Brassicaceae) exhibits seed dimorphism as a bet-hedging strategy - producing both a less dormant mucilaginous (M+) seed morph and a more dormant non-mucilaginous (NM) seed morph. Here, we compared de novo and reference-genome based transcriptome assemblies to investigate Ae. arabicum seed dimorphism and to evaluate the reference-free versus -dependent approach for identifying differentially expressed genes (DEGs). RESULTS: A de novo transcriptome assembly was generated using sequences from M+ and NM Ae. arabicum dry seed morphs. The transcripts of the de novo assembly contained 63.1% complete Benchmarking Universal Single-Copy Orthologs (BUSCO) compared to 90.9% for the transcripts of the reference genome. DEG detection used the strict consensus of three methods (DESeq2, edgeR and NOISeq). Only 37% of 1533 differentially expressed de novo assembled transcripts paired with 1876 genome-derived DEGs. Gene Ontology (GO) terms distinguished the seed morphs: the terms translation and nucleosome assembly were overrepresented in DEGs higher in abundance in M+ dry seeds, whereas terms related to mRNA processing and transcription were overrepresented in DEGs higher in abundance in NM dry seeds. DEGs amongst these GO terms included ribosomal proteins and histones (higher in M+), RNA polymerase II subunits and related transcription and elongation factors (higher in NM). Expression of the inferred DEGs and other genes associated with seed maturation (e.g. those encoding late embryogenesis abundant proteins and transcription factors regulating seed development and maturation such as ABI3, FUS3, LEC1 and WRI1 homologs) were put in context with Arabidopsis thaliana seed maturation and indicated that M+ seeds may desiccate and mature faster than NM. The 1901 transcriptomic DEG set GO-terms had almost 90% overlap with the 2191 genome-derived DEG GO-terms. CONCLUSIONS: Whilst there was only modest overlap of DEGs identified in reference-free versus -dependent approaches, the resulting GO analysis was concordant in both approaches. The identified differences in dry seed transcriptomes suggest mechanisms underpinning previously identified contrasts between morphology and germination behaviour of M+ and NM seeds.

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.

Long days induce adaptive secondary dormancy in the seeds of the Mediterranean plant Aethionema arabicum.

Secondary dormancy is an adaptive trait that increases reproductive success by aligning seed germination with permissive conditions for seedling establishment. Aethionema arabicum is an annual plant and member of the Brassicaceae that grows in environments characterized by hot and dry summers. Aethionema arabicum seeds may germinate in early spring when seedling establishment is permissible. We demonstrate that long-day light regimes induce secondary dormancy in the seeds of Aethionema arabicum (CYP accession), repressing germination in summer when seedling establishment is riskier. Characterization of mutants screened for defective secondary dormancy demonstrated that RGL2 mediates repression of genes involved in gibberellin (GA) signaling. Exposure to high temperature alleviates secondary dormancy, restoring germination potential. These data are consistent with the hypothesis that long-day-induced secondary dormancy and its alleviation by high temperatures may be part of an adaptive response limiting germination to conditions permissive for seedling establishment in spring and autumn.

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.

Fern genomes elucidate land plant evolution and cyanobacterial symbioses.

Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of 'core leptosporangiates' and one specific to Azolla. One fern-specific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N2-fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Comprehensive Genome-Wide Classification Reveals That Many Plant-Specific Transcription Factors Evolved in Streptophyte Algae.

Plant genomes encode many lineage-specific, unique transcription factors. Expansion of such gene families has been previously found to coincide with the evolution of morphological complexity, although comparative analyses have been hampered by severe sampling bias. Here, we make use of the recently increased availability of plant genomes. We have updated and expanded previous rule sets for domain-based classification of transcription associated proteins (TAPs), comprising transcription factors and transcriptional regulators. The genome-wide annotation of these protein families has been analyzed and made available via the novel TAPscan web interface. We find that many TAP families previously thought to be specific for land plants actually evolved in streptophyte (charophyte) algae; 26 out of 36 TAP family gains are inferred to have occurred in the common ancestor of the Streptophyta (uniting the land plants-Embryophyta-with their closest algal relatives). In contrast, expansions of TAP families were found to occur throughout streptophyte evolution. 17 out of 76 expansion events were found to be common to all land plants and thus probably evolved concomitant with the water-to-land-transition.