Biology subjects

Köhler, C.

Publications and source records attributed to Köhler, C..

12 recordsLinked to original sources

bioRxiv2026

SirenScan reveals the conserved presence of siren RNAs and their evolutionary diversification across angiosperms

O_LISiren RNAs are exceptionally abundant reproductive small interfering RNAs (siRNAs), yet whether they represent a conserved feature of angiosperm reproduction has remained unclear because siren loci lack a standardized definition and identification strategy. C_LIO_LIWe developed SirenScan, a computational framework that integrates cumulation-based and density-based analyses to identify siren loci from small RNA sequencing data. Using SirenScan, we systematically compared ovule and vegetative tissues across representative angiosperm lineages, including the early-diverging angiosperm Nymphaea colorata. C_LIO_LIWe demonstrate that the presence of siren RNAs is a conserved feature of angiosperm ovules, although the degree of expression dominance varies substantially among species. Siren loci are consistently enriched within transposable elements (TEs), while the associated TE superfamilies and cis-regulatory motifs exhibit lineage-specific diversification. In Brassicaceae, siren loci share conserved CLASSY3-associated sequence motifs, whereas other angiosperm lineages display distinct motif compositions, suggesting evolutionary diversification of Polymerase IV targeting mechanisms. C_LIO_LIOur findings establish siren RNAs as a conserved component of the angiosperm reproductive small RNA landscape and support a model in which conserved RNA-directed DNA methylation machinery is coupled with lineage-specific regulatory mechanisms and transposon landscapes to shape siren locus evolution. SirenScan provides a robust and standardized framework for comparative studies of siren RNA biology and reproductive epigenomics across plant species. C_LI

plant biology↗

bioRxiv2025

Samplify: A versatile tool for image-based segmentation and annotation of seed abortion phenotypes

Automated seed phenotyping has wide applications in research and agriculture and relies on easy-to-use platforms and pipelines. Seed phenotyping in the model species Arabidopsis thaliana poses a significant challenge due to the large number of tiny seeds produced by individual plants, which are difficult to manually separate and count. Manual counting methods are time-consuming and prone to user bias, particularly for subtle phenotypic changes. To address these limitations, we developed Samplify, a scalable, automated pipeline for seed segmentation and classification. By integrating classical image processing techniques with Metas Segment Anything Model (SAM), Samplify effectively segments Arabidopsis seeds, even in dense clusters where conventional methods fail. To demonstrate its versatility, we quantified the seed abortion occurring in interploidy crossings in Arabidopsis, often referred to as triploid block. Samplify includes a Random Forest classifier trained on a set of computed seed shape features that enables the categorization of seeds into normal, partially aborted, and fully aborted seeds, automating the manual classification process. The tool, designed as a command-line application, significantly reduces manual annotation workload. Our validation across multiple datasets demonstrates high segmentation and classification reliability, making Samplify a valuable resource for the plant research community.

plant biology↗

bioRxiv2025

Maternal control of RNA decay safeguards embryo development

As in mammals, the plant embryo is surrounded by maternal tissues that provide protection from the external environment. In angiosperms, a double fertilization process results in the formation of a diploid embryo and a triploid endosperm, both of which develop within maternal sporophytic integuments. Thus, the seed of angiosperms is a combined structure of three genetically distinct components: embryo, endosperm, and maternal integuments. It has long been assumed that the maternal plant influences embryo development, but there is limited molecular evidence for a direct pathway through which the maternal sporophyte aYects embryogenesis. Here we show that secondary small interfering RNAs (siRNAs) generated upon exosome impairment lead to embryo abortion through a maternal sporophytic eYect. Depletion of the core subunit of the RNA-processing exosome RRP45B (CER7) causes globular embryo arrest connected to ectopic post-transcriptional gene silencing (PTGS). Seed coat expression of CER7 suppresses seed abortion, demonstrating a maternal sporophytic control of embryo development through an RNA decay safeguard pathway. Our data support a model in which a primary siRNA trigger originates in the maternal integuments of the cer7 seed coat and drives PTGS amplification in reproductive tissues after fertilization, ultimately leading to seed abortion. In addition, our genetic and molecular data suggest that overloading of AGO1 with siRNAs impairs miRNA function, likely leading to embryo arrest. Our data highlight the complex interplay between maternal and embryonic gene regulation, reinforcing the importance of controlled RNA decay in plant development.

plant biology↗

bioRxiv2024

Epigenetic and transcriptional consequences of chemically induced transposon mobilization in the endosperm

Genomic imprinting, an epigenetic phenomenon leading to parent-of-origin-specific gene expression, has independently evolved in the endosperm of flowering plants and the placenta of mammals--tissues crucial for nurturing embryos. While transposable elements (TEs) frequently colocalize with imprinted genes and are implicated in imprinting establishment, direct investigations of the impact of de novo TE transposition on genomic imprinting remain scarce. In this study, we explored the effects of chemically induced transposition of the Copia element ONSEN on genomic imprinting in Arabidopsis thaliana. Through the combination of chemical TE mobilization and doubled haploid induction, we generated a line with 40 new ONSEN copies. Our findings reveal a preferential targeting of maternally expressed genes (MEGs) for transposition, aligning with the colocalization of H2A.Z and H3K27me3 in MEGs-- both previously identified as promoters of ONSEN insertions. Additionally, we demonstrate that chemically-induced DNA hypomethylation induces global transcriptional deregulation in the endosperm, leading to the breakdown of MEG imprinting. This study provides insights into the consequences of chemically induced TE remobilization in the endosperm, underscoring the need for cautious interpretation of the connection between TEs and genomic imprinting.

plant biology↗

bioRxiv2023

Updated phylogeny and protein structure predictions revise the hypothesis on the origin of MADS-box transcription factors in land plants

MADS-box transcription factors (TFs) are broadly present in eukaryotes. Varying by domain architecture, MADS-box TFs in land plants are categorized into Type I (M-type) and Type II (MIKC-type). For about twenty years, Type I and II genes were considered orthologous to the SRF and MEF2 genes in animals, respectively, presumably originating from a duplication before the divergence of eukaryotes. Here, we exploited the increasing eukaryotic MADS-box sequences and reassessed their evolution. While supporting the ancient duplication giving rise to SRF- and MEF2-types, we found that Type I and II genes originated from the MEF2-type genes through another duplication in the most recent common ancestor (MRCA) of land plants. Protein structures predicted by AlphaFold2 and OmegaFold support our phylogenetic analyses, with plant Type I and II TFs resembling the MEF2-type structure, rather than SRFs. We hypothesize that the ancestral SRF-type TFs got lost in the MRCA of Archaeplastida (the kingdom Plantae sensu lato). The retained MEF2-type TFs acquired a Keratin-like domain and became MIKC-type upon the evolution of Streptophyta. Subsequently in the MRCA of land plants, M-type TFs evolved from a duplicated MIKC-type precursor through loss of the Keratin-like domain, leading to the Type I clade. Both Type I and II TFs largely expanded and functionally differentiated in concert with the increasing complexity of land plant body architecture. We attribute the adaptation to the terrestrial environment partly to the divergence among MEF2-type MADS-box genes and the repetitive recruitment of these originally stress-responsive TFs into developmental programs, especially those underlying reproduction.

plant biology↗

bioRxiv2021

Chemically-induced epimutagenesis allows bypassing reproductive barriers in hybrid seeds

The "triploid block" prevents interploidy hybridizations in flowering plants, and is characterized by failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed imprinted genes (PEGs) that are up-regulated in the tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We have identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of "epimutagenized" plants, which may allow the identification of epialleles involved in the triploid block response in future studies. Finally, we demonstrate that chemically-induced epimutagenesis allows bypassing hybridization barriers in crosses between different Capsella species, thus potentially emerging as a novel strategy for producing triploids and interspecific hybrids with high agronomical interest. One sentence summaryGenome-wide loss of DNA methylation induced by 5-Azacytidine allows bypassing interploidy and interspecific hybridization barriers in Arabidopsis and Capsella.

plant biology↗

bioRxiv2021

Endosperm evolution by duplicated and neofunctionalized Type I MADS-box transcription factors

MADS-box transcription factors (TFs) are present in nearly all major eukaryotic groups. They are divided into Type I and Type II that differ in domain structure, functional roles, and rates of evolution. In flowering plants, major evolutionary innovations like flowers, ovules and fruits have been closely connected to Type II MADS-box TFs. The role of Type I MADS-box TFs in angiosperm evolution remains to be identified. Here, we show that the formation of angiosperm-specific Type I MADS-box clades of M{gamma} and M{gamma}-interacting M genes (M*) can be tracked back to the ancestor of all angiosperms. Angiosperm-specific M{gamma} and M* genes were preferentially expressed in the endosperm, consistent with their proposed function as heterodimers in the angiosperm-specific embryo-nourishing endosperm tissue. We propose that duplication and diversification of Type I MADS-genes underpins the evolution of the endosperm, a developmental innovation closely connected to the origin and success of angiosperms.

plant biology↗

bioRxiv2020

Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy

Polycomb Repressive Complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) and methylation of histone 3 on lysine 9 (H3K9me) are two repressive epigenetic modifications that are typically localized in distinct regions of the genome. For reasons unknown, however, they co-occur in some organisms and special tissue types. In this study, we show that maternal alleles marked by H3K27me3 in the Arabidopsis endosperm were targeted by the H3K27me3 demethylase REF6 and became activated during germination. In contrast, maternal alleles marked by H3K27me3, H3K9me2, and CHGm were protected from REF6 targeting and remained silenced. Our study unveils that combinations of different repressive epigenetic modifications time a key adaptive trait by modulating access of REF6.

molecular biology↗

bioRxiv2020

Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block

Hybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. Mutation in NRPD1, encoding the largest subunit of the plant-specific RNA Polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. In this study, we found that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While nrpd1 is able to suppress in the first homozygous generation, mutants in RDR2, NRPE1, and DRM2 require at least one additional round of inbreeding to exert a suppressive effect. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3. Our data thus reveal that loss of RdDM function differs in its effect in early and late generations and that Pol IV acts at an early stage of triploid block establishment. One-sentence summaryInbreeding of mutants impaired in RdDM components transgenerationally enhanced their ability to suppress the triploid block.

genetics↗

bioRxiv2020

Hybrid seed incompatibility in Capsella is connected to chromatin condensation defects in the endosperm

Hybridization of closely related plant species is frequently connected to endosperm arrest and seed failure, for reasons that remain to be identified. In this study, we investigated the molecular events accompanying seed failure in hybrids of the closely related species pair Capsella rubella and C. grandiflora. Mapping of QTLs for the underlying cause of hybrid incompatibility in Capsella revealed three QTLs that were close to pericentromeric regions. This prompted us to investigate whether there are specific changes in heterochromatin associated with interspecific hybridizations. Indeed, we found that chromatin was less condensed in the endosperm, while the embryo was not affected. Loss of chromosome condensation was connected with a strong loss of CHG and CHH methylation and mitotic abnormalities. Genome-wide sequencing of hybrid endosperm revealed that the chromosome loss was random and was likely a consequence of reduced chromatin condensation. Consistent with reduced DNA methylation in hybrid endosperm, we found a disproportionate deregulation of genes located close to pericentromeric regions. Among those deregulated genes there were many potential targets of the AGAMOUS-LIKE transcription factor PHERES1, suggesting that reduced DNA methylation allows PHERES1 to hyperactivate its targets. Since the identified QTLs were also associated with pericentromeric regions, we conclude that relaxation of heterochromatin in response to interspecies hybridization exposes and activates loci leading to hybrid seed failure.

evolutionary biology↗

bioRxiv2018

Genetic basis and timing of a major mating system shift in Capsella

Shifts from outcrossing to self-fertilisation have occurred repeatedly in many different lineages of flowering plants, and often involve the breakdown of genetic outcrossing mechanisms. In the Brassicaceae, self-incompatibility (SI) allows plants to ensure outcrossing by recognition and rejection of self-pollen on the stigma. This occurs through the interaction of female and male specificity components, consisting of a pistil based receptor and a pollen-coat protein, both of which are encoded by tightly linked genes at the S-locus. When benefits of selfing are higher than costs of inbreeding, theory predicts that loss-of-function mutations in the male (pollen) SI component should be favoured, especially if they are dominant. However, it remains unclear whether mutations in the male component of SI are predominantly responsible for shifts to self-compatibility, and testing this prediction has been difficult due to the challenges of sequencing the highly polymorphic and repetitive ~100 kbp S-locus. The crucifer genus Capsella offers an excellent opportunity to study multiple transitions from outcrossing to self-fertilization, but so far, little is known about the genetic basis and timing of loss of SI in the self-fertilizing diploid Capsella orientalis. Here, we show that loss of SI in C. orientalis occurred within the past 2.6 Mya and maps as a dominant trait to the S-locus. Using targeted long-read sequencing of multiple complete S-haplotypes, we identify a frameshift deletion in the male specificity gene SCR that is fixed in C. orientalis, and we confirm loss of male SI specificity. We further analyze RNA sequencing data to identify a conserved, S-linked small RNA (sRNA) that is predicted to cause dominance of self-compatibility. Our results suggest that degeneration of pollen SI specificity in dominant S-alleles is important for shifts to self-fertilization in the Brassicaceae.\n\nAuthor SummaryAlready Darwin was fascinated by the widely varying modes of plant reproduction. The shift from outcrossing to self-fertilization is considered one of the most frequent evolutionary transitions in flowering plants, yet we still know little about the genetic basis of these shifts. In the Brassicaceae, outcrossing is enforced by a self-incompatibility (SI) system that enables the recognition and rejection of self pollen. This occurs through the action of two tightly linked genes at the S-locus, that encode a receptor protein located on the stigma (female component) and a pollen ligand protein (male component), respectively. Nevertheless, SI has frequently been lost, and theory predicts that mutations in the male component should have an advantage during the loss of SI, especially if they are dominant. To test this hypothesis, we mapped the loss of SI in a selfing species from the genus Capsella, a model system for evolutionary genomics. We found that loss of SI mapped to the S-locus, which harbored a dominant loss-of-function mutation in the male SI protein, and as expected, we found that male specificity was indeed lost in C. orientalis. Our results suggest that transitions to selfing often involve parallel genetic changes.

evolutionary biology↗

bioRxiv2018

Epigenetic signatures associated with imprinted paternally-expressed genes in the Arabidopsis endosperm

BackgroundImprinted genes are epigenetically modified during gametogenesis and maintain the established epigenetic signatures after fertilization, causing parental-specific gene expression.\n\nResultsIn this study, we show that imprinted paternally-expressed genes (PEGs) in the Arabidopsis endosperm are marked by an epigenetic signature of Polycomb Repressive Complex2 (PRC2)-mediated H3K27me3 together with heterochromatic H3K9me2 and CHG methylation, which specifically mark the silenced maternal alleles of PEGs. The co-occurrence of H3K27me3 and H3K9me2 on defined loci in the endosperm drastically differs from the strict separation of both pathways in vegetative tissues, revealing tissue-specific employment of repressive epigenetic pathways in plants. Based on the presence of this epigenetic signature on maternal alleles we were able to predict known PEGs at high accuracy and identified several new PEGs that we confirmed using INTACT-based transcriptomes generated in this study.\n\nConclusionsThe presence of the three repressive epigenetic marks, H3K27me3, H3K9me2, and CHG methylation on the maternal alleles in the endosperm serves as a specific epigenetic signature that allows to predict genes with parental-specific gene expression. Our study reveals that there are substantially more PEGs than previously identified, indicating that paternal-specific gene expression is of higher functional relevance than currently estimated. The combined activity of PRC2-mediated H3K27me3 together with the heterochromatic H3K9me3 has also been reported to silence the maternal Xist locus in mammalian preimplantation embryos, suggesting convergent employment of both pathways during the evolution of genomic imprinting.

genetics↗