Structural evidence for MADS-box type I family expansion seen in new assemblies of Arabidopsis arenosa and A. lyrata.

Arabidopsis thaliana diverged from A. arenosa and A. lyrata at least 6 million years ago. The three species differ by genome-wide polymorphisms and morphological traits. The species are to a high degree reproductively isolated, but hybridization barriers are incomplete. A special type of hybridization barrier is based on the triploid endosperm of the seed, where embryo lethality is caused by endosperm failure to support the developing embryo. The MADS-box type I family of transcription factors is specifically expressed in the endosperm and has been proposed to play a role in endosperm-based hybridization barriers. The gene family is well known for its high evolutionary duplication rate, as well as being regulated by genomic imprinting. Here we address MADS-box type I gene family evolution and the role of type I genes in the context of hybridization. Using two de-novo assembled and annotated chromosome-level genomes of A. arenosa and A. lyrata ssp. petraea we analyzed the MADS-box type I gene family in Arabidopsis to predict orthologs, copy number, and structural genomic variation related to the type I loci. Our findings were compared to gene expression profiles sampled before and after the transition to endosperm cellularization in order to investigate the involvement of MADS-box type I loci in endosperm-based hybridization barriers. We observed substantial differences in type-I expression in the endosperm of A. arenosa and A. lyrata ssp. petraea, suggesting a genetic cause for the endosperm-based hybridization barrier between A. arenosa and A. lyrata ssp. petraea.

Spatial and temporal regulation of parent-of-origin allelic expression in the endosperm.

Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Regional compartmentalization has been shown both in early and late endosperm development, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and the investigation of domain-specific functions have been hampered by the inaccessibility of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here, we used fluorescence-activated nuclear sorting (FANS) of nuclear targeted GFP fluorescent genetic markers to capture parental-specific allelic expression from different developmental stages and specific endosperm domains. This approach allowed us to successfully identify differential genomic imprinting with temporal and spatial resolution. We used a systematic approach to report temporal regulation of imprinted genes in the endosperm, as well as region-specific imprinting in endosperm domains. Analysis of our data identified loci that are spatially differentially imprinted in one domain of the endosperm, while biparentally expressed in other domains. These findings suggest that the regulation of genomic imprinting is dynamic and challenge the canonical mechanisms for genomic imprinting.

Genetic variation and temperature affects hybrid barriers during interspecific hybridization.

Genomic imprinting regulates parent-specific transcript dosage during seed development and is mainly confined to the endosperm. Elucidation of the function of many imprinted genes has been hampered by the lack of corresponding mutant phenotypes, and the role of imprinting is mainly associated with genome dosage regulation or allocation of resources. Disruption of imprinted genes has also been suggested to mediate endosperm-based post-zygotic hybrid barriers depending on genetic variation and gene dosage. Here, we have analyzed the conservation of a clade from the MADS-box type I class transcription factors in the closely related species Arabidopsis arenosa, A. lyrata, and A. thaliana, and show that AGL36-like genes are imprinted and maternally expressed in seeds of Arabidopsis species and in hybrid seeds between outbreeding species. In hybridizations between outbreeding and inbreeding species the paternally silenced allele of the AGL36-like gene is reactivated in the hybrid, demonstrating that also maternally expressed imprinted genes are perturbed during hybridization and that such effects on imprinted genes are specific to the species combination. Furthermore, we also demonstrate a quantitative effect of genetic diversity and temperature on the strength of the post-zygotic hybridization barrier. Markedly, a small decrease in temperature during seed development increases the survival of hybrid F1 seeds, suggesting that abiotic and genetic parameters play important roles in post-zygotic species barriers, pointing at evolutionary scenarios favoring such effects. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA562212. All sequences generated in this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/) with project number PRJNA562212.

Regulation of Parent-of-Origin Allelic Expression in the Endosperm.

Genomic imprinting is an epigenetic phenomenon established in the gametes prior to fertilization that causes differential expression of parental alleles, mainly in the endosperm of flowering plants. The overlap between previously identified panels of imprinted genes is limited. To investigate imprinting, we used high-resolution sequencing data acquired with sequence-capture technology. We present a bioinformatics pipeline to assay parent-of-origin allele-specific expression and report more than 300 loci with parental expression bias in Arabidopsis (Arabidopsis thaliana). In most cases, the level of expression from maternal and paternal alleles was not binary, instead supporting a differential dosage hypothesis for the evolution of imprinting in plants. To address imprinting regulation, we systematically employed mutations in regulative epigenetic pathways suggested to be major players in the process. We established the mechanistic mode of imprinting for more than 50 loci regulated by DNA methylation and Polycomb-dependent histone methylation. However, the imprinting patterns of most genes were not affected by these mechanisms. To this end, we also demonstrated that the RNA-directed DNA methylation pathway alone does not substantially influence imprinting patterns, suggesting that more complex epigenetic pathways regulate most of the identified imprinted genes.

Endosperm-based hybridization barriers explain the pattern of gene flow between Arabidopsis lyrata and Arabidopsis arenosa in Central Europe.

Based on the biological species concept, two species are considered distinct if reproductive barriers prevent gene flow between them. In Central Europe, the diploid species Arabidopsis lyrata and Arabidopsis arenosa are genetically isolated, thus fitting this concept as "good species." Nonetheless, interspecific gene flow involving their tetraploid forms has been described. The reasons for this ploidy-dependent reproductive isolation remain unknown. Here, we show that hybridization between diploid A. lyrata and A. arenosa causes mainly inviable seed formation, revealing a strong postzygotic reproductive barrier separating these two species. Although viability of hybrid seeds was impaired in both directions of hybridization, the cause for seed arrest differed. Hybridization of A. lyrata seed parents with A. arenosa pollen donors resulted in failure of endosperm cellularization, whereas the endosperm of reciprocal hybrids cellularized precociously. Endosperm cellularization failure in both hybridization directions is likely causal for the embryo arrest. Importantly, natural tetraploid A. lyrata was able to form viable hybrid seeds with diploid and tetraploid A. arenosa, associated with the reestablishment of normal endosperm cellularization. Conversely, the defects of hybrid seeds between tetraploid A. arenosa and diploid A. lyrata were aggravated. According to these results, we hypothesize that a tetraploidization event in A. lyrata allowed the production of viable hybrid seeds with A. arenosa, enabling gene flow between the two species.

The IDA/IDA-LIKE and PIP/PIP-LIKE gene families in Arabidopsis: phylogenetic relationship, expression patterns, and transcriptional effect of the PIPL3 peptide.

Peptide ligands play crucial roles in the life cycle of plants by modulating the innate immunity against pathogens and regulating growth and developmental processes. One well-studied example is INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which controls floral organ abscission and lateral root emergence in Arabidopsis thaliana. IDA belongs to a family of five additional IDA-LIKE (IDL) members that have all been suggested to be involved in regulation of Arabidopsis development. Here we present three novel members of the IDL subfamily and show that two of them are strongly and rapidly induced by different biotic and abiotic stresses. Furthermore, we provide data that the recently identified PAMP-INDUCED SECRETED PEPTIDE (PIP) and PIP-LIKE (PIPL) peptides, which show similarity to the IDL and C-TERMINALLY ENCODED PEPTIDE (CEP) peptides, are not only involved in innate immune response in Arabidopsis but are also induced by abiotic stress. Expression patterns of the IDA/IDL and PIP/PIPL genes were analysed using in silico data, qRT-PCR and GUS promoter lines. Transcriptomic responses to PIPL3 peptide treatment suggested a role in regulation of biotic stress responses and cell wall modification.