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.

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.

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).

Between a rock and a hard place: adaptive sensing and site-specific dispersal.

Environmental variability can lead to dispersal: why stay put if it is better elsewhere? Without clues about local conditions, the optimal strategy is often to disperse a set fraction of offspring. Many habitats contain environmentally differing sub-habitats. Is it adaptive for individuals to sense in which sub-habitat they find themselves, using environmental clues, and respond plastically by altering the dispersal rates? This appears to be done by some plants which produce dimorphic seeds with differential dispersal properties in response to ambient temperature. Here we develop a mathematical model to show, that in highly variable environments, not only does sensing promote plasticity of dispersal morph ratio, individuals who can sense their sub-habitat and respond in this way have an adaptive advantage over those who cannot. With a rise in environmental variability due to climate change, our understanding of how natural populations persist and respond to changes has become crucially important.

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.

Aethionema arabicum: a novel model plant to study the light control of seed germination.

The timing of seed germination is crucial for seed plants and is coordinated by internal and external cues, reflecting adaptations to different habitats. Physiological and molecular studies with lettuce and Arabidopsis thaliana have documented a strict requirement for light to initiate germination and identified many receptors, signaling cascades, and hormonal control elements. In contrast, seed germination in several other plants is inhibited by light, but the molecular basis of this alternative response is unknown. We describe Aethionema arabicum (Brassicaceae) as a suitable model plant to investigate the mechanism of germination inhibition by light, as this species has accessions with natural variation between light-sensitive and light-neutral responses. Inhibition of germination occurs in red, blue, or far-red light and increases with light intensity and duration. Gibberellins and abscisic acid are involved in the control of germination, as in Arabidopsis, but transcriptome comparisons of light- and dark-exposed A. arabicum seeds revealed that, upon light exposure, the expression of genes for key regulators undergo converse changes, resulting in antipodal hormone regulation. These findings illustrate that similar modular components of a pathway in light-inhibited, light-neutral, and light-requiring germination among the Brassicaceae have been assembled in the course of evolution to produce divergent pathways, likely as adaptive traits.

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.

Dispersal biophysics and adaptive significance of dimorphic diaspores in the annual Aethionema arabicum (Brassicaceae).

Heteromorphic diaspores (fruits and seeds) are an adaptive bet-hedging strategy to cope with spatiotemporally variable environments, particularly fluctuations in favourable temperatures and unpredictable precipitation regimes in arid climates. We conducted comparative analyses of the biophysical and ecophysiological properties of the two distinct diaspores (mucilaginous seed (M+ ) vs indehiscent (IND) fruit) in the dimorphic annual Aethionema arabicum (Brassicaceae), linking fruit biomechanics, dispersal aerodynamics, pericarp-imposed dormancy, diaspore abscisic acid (ABA) concentration, and phenotypic plasticity of dimorphic diaspore production to its natural habitat and climate. Two very contrasting dispersal mechanisms of the A. arabicum dimorphic diaspores were revealed. Dehiscence of large fruits leads to the release of M+ seed diaspores, which adhere to substrata via seed coat mucilage, thereby preventing dispersal (antitelechory). IND fruit diaspores (containing nonmucilaginous seeds) disperse by wind or water currents, promoting dispersal (telechory) over a longer range. The pericarp properties confer enhanced dispersal ability and degree of dormancy on the IND fruit morph to support telechory, while the M+ seed morph supports antitelechory. Combined with the phenotypic plasticity to produce more IND fruit diaspores in colder temperatures, this constitutes a bet-hedging survival strategy to magnify the prevalence in response to selection pressures acting over hilly terrain.

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.