Vojtěch Čermák

Summary paragraph: Imprinted genes do not act alone to shape seed development, but as a complex network — just like any other gene. Yet, the molecular context in which they are embedded, i.e. their gene network, remains largely understudied. To address this knowledge gap, we characterized the imprintome of Arabidopsis arenosa at the species-level and used gene regulatory network analyses. We show that genomic imprinting preferentially affects only a few pathways, offering candidates for dosage sensitive processes and the molecular arena of parental conflict. In these pathways, some imprinted genes act as hub genes, among which NRPE1 , highlighting the importance of epigenetics in endosperm development. The interaction between parental genomes was rather one-sided: paternally expressed regulators preferentially targeted PEGs, while maternally expressed regulators targeted both PEGs and MEGs indiscriminately. This aligns with a self-promoting paternal influence and a maternal buffer under a parental conflict scenario. Shared paternal and maternal regulation of downstream targets was nevertheless common, and we reveal novel molecular interactions between imprinted regulators. Overall, our work shows how genomic imprinting may act as a molecular means for parental conflict, but also highlights the importance of parental molecular coordination in endosperm development.

SUMMARY: Polyploidy is a key evolutionary force in plants; among its many consequences, hybridization between diploids and polyploids is restricted due to the “triploid block”. While the molecular mechanisms of this postzygotic barrier are extensively studied in the model species Arabidopsis thaliana , our understanding of the triploid block in natural systems remains limited. Here, we investigated the transcriptome of failing triploid seeds in Arabidopsis arenosa , a close relative of A. thaliana with diploid and autotetraploid populations meeting in nature. We also identified A. arenosa imprinted genes. Triploid seeds showed preferential misregulation of imprinted genes, which parallels the parent-of-origin features of the triploid block. Tissue-specific transcriptomic analyses revealed pathogen defense-like response being recurrently affected in the endosperm and seed coat. This pathway is commonly misregulated in all species with triploid seed transcriptomes studied to date. The associated genes however are likely more involved in cell-cell signaling rather than pathogen defense per se . Altogether, this study depicts a thorough molecular landscape of the triploid block for the first time in natural systems. Combining data on the understudied maternal excess transcriptome, a new imprintome, a tissue-specific focus, and a cross-species comparison, this study also advances our understanding of the triploid block and seed development.

In plants, an essential component of the RNA Polymerase II (Pol II) complex, SPT6L, contains a unique C-terminal ARGONAUTE binding domain - AGO-hook. Although it has been recently shown that SPT6L is also part of the Pol V complex, where its AGO-hook might participate in the RNA-directed DNA methylation pathway, several lines of evidence suggest that the AGO-hook should also have a function in the Pol II complex. Here we demonstrate that in Arabidopsis thaliana , SPT6L recruits AGO4 via the AGO-hook domain, independently of Pol V. Using direct RNA sequencing, we show that this interaction is connected with guided deposition of 5-methylcytosine (m ⁵ C) on Pol II transcripts. The role of AGO4 in m ⁵ C guidance was further supported by increased methylation of GFP transcripts after their targeting by sRNAs in tobacco BY-2 cells. Removing the SPT6L AGO-hook ( spt6lΔAh ) in A. thaliana resulted in the hypomethylation of specific m ⁵ C sites, mimicking even stronger RNA hypomethylation observed in the ago4 mutant. Native elongating transcript sequencing revealed that impaired targeting of m ⁵ C methylation in the spt6lΔAh mutant was accompanied by Pol II stalling downstream of hypomethylated cytosines. Furthermore, this stalling appeared to be associated with premature transcription termination, which we also detected in published transcriptomes of mutants lacking AGO4, as well as TRM4B that is the major RNA m ⁵ C methyltransferase. Our findings uncover a novel “guide-and-modify” mechanism in which SPT6L integrates sRNA-directed epitranscriptomic marking with transcription elongation, ensuring proper mRNA termination.

Purifying selection is expected to prevent the accumulation of transposable elements (TEs) within their host, especially when located in and around genes and if affected by epigenetic silencing. However, positive selection may favor the spread of TEs, causing genomic imprinting under parental conflict, as genomic imprinting allows parent-specific influence over resource accumulation to the progeny. Concomitantly, the number and frequency of TE insertions in natural populations are conditioned by demographic events. In this study, we aimed to test how demography and selective forces interact to affect the accumulation of TEs around genes, depending on their epigenetic silencing, with a particular focus on imprinted genes. To this aim, we compared the frequency and distribution of TEs in Arabidopsis lyrata from Europe and North America. Generally, we found that TE insertions showed a lower frequency when they were inserted in or near genes, especially TEs targeted by epigenetic silencing, suggesting purifying selection at work. We also found that many TEs were lost or got fixed in North American populations during the colonization and the postglacial range expansion from refugia of the species in North America, as well as during the transition to selfing, suggesting a potential “TE load.” Finally, we found that silenced TEs increased in frequency and even tended to reach fixation when they were linked to imprinted genes. We conclude that in A. lyrata, genomic imprinting has spread in natural populations through demographic events and positive selection acting on silenced TEs, potentially under a parental conflict scenario.

RNA-directed DNA methylation (RdDM) is driven by small RNAs (sRNAs) complementary to the nascent transcript of RNA polymerase V (Pol V). sRNAs associated with ARGONAUTE (AGO) proteins are tethered to Pol V mainly by the AGO-hook domain of its subunit NRPE1. We found, by in silico analyses, that Pol V strongly colocalizes on chromatin with another AGO-hook protein, SPT6-like (SPT6L), which is a known essential transcription elongation factor of Pol II. Our phylogenetic analysis revealed that SPT6L acquired its AGO-binding capacity already in the most basal streptophyte algae, even before the emergence of Pol V, suggesting that SPT6L might be a driving force behind the RdDM evolution. Since its emergence, SPT6L with the AGO-hook represents the only conserved SPT6 homolog in Viridiplantae, implying that the same protein is involved in both Pol II and Pol V complexes. To better understand the role of SPT6L in the Pol V complex, we characterized genomic loci where these two colocalize and uncovered that DNA methylation there is more dynamic, driven by higher levels of sRNAs often from non-canonical RdDM pathways and more dependent on chromatin modifying and remodeling proteins like MORC. Pol V loci with SPT6L are highly depleted in helitrons but enriched in gene promoters for which locally and temporally precise methylation is necessary. In view of these results, we discuss potential roles of multiple AGO-hook domains present in the Pol V complex and speculate that SPT6L mediates de novo methylation of naïve loci by interconnecting Pol II and Pol V activities.

RNA-directed DNA methylation (RdDM) is driven by small RNAs (sRNAs) complementary to the nascent transcript of RNA polymerase V (Pol V). sRNAs associated with ARGONAUTE (AGO) proteins are tethered to Pol V mainly by the AGO-hook domain of its subunit NRPE1. We found, by in silico analyses, that Pol V strongly colocalizes on chromatin with another AGO-hook protein, SPT6-like (SPT6L), which is a known essential transcription elongation factor of Pol II. Our phylogenetic analysis revealed that SPT6L acquired its AGO-binding capacity already in the most basal streptophyte algae, even before the emergence of Pol V, suggesting that SPT6L might be a driving force behind the RdDM evolution. Since its emergence, SPT6L with the AGO-hook represents the only conserved SPT6 homolog in Viridiplantae , implying that the same protein is involved in both Pol II and Pol V complexes. To better understand the role of SPT6L in the Pol V complex, we characterized genomic loci where these two colocalize and uncovered that DNA methylation there is more dynamic, driven by higher levels of sRNAs often from non-canonical RdDM pathways and more dependent on chromatin modifying and remodeling proteins like MORC. Pol V loci with SPT6L are highly depleted in helitrons but enriched in gene promoters for which locally and temporally precise methylation is necessary. In view of these results, we discuss potential roles of multiple AGO-hook domains present in the Pol V complex and speculate that SPT6L mediates de novo methylation of naïve loci by interconnecting Pol II and Pol V activities.

Heat stress (HS) is a major abiotic stress that negatively impacts crop yields across the globe. Plants respond to elevated temperatures by changing gene expression, mediated by transcription factors (TFs) functioning to enhance HS tolerance. The involvement of Group I bZIP TFs in the heat stress response (HSR) is not known. In this study, bZIP18 and bZIP52 were investigated for their possible role in the HSR. Localization experiments revealed their nuclear accumulation following heat stress, which was found to be triggered by dephosphorylation. Both TFs were found to possess two motifs containing serine residues that are candidates for phosphorylation. These motifs are recognized by 14–3–3 proteins, and bZIP18 and bZIP52 were found to bind 14–3–3 ε, the interaction of which sequesters them to the cytoplasm. Mutation of both residues abolished 14–3–3 ε interaction and led to a strict nuclear localization for both TFs. RNA-seq analysis revealed coordinated downregulation of several metabolic pathways including energy metabolism and translation, and upregulation of numerous lncRNAs in particular. These results support the idea that bZIP18 and bZIP52 are sequestered to the cytoplasm under control conditions, and that heat stress leads to their re-localization to nuclei, where they jointly regulate gene expression.

Background: Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert.Results: We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to theCaMV 35Spromoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases.Conclusions: Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

Here we provide an overview of procedures for long-term cultivation, phenotyping, genotyping, and genetic transformation of cell cultures of tobacco cell lines BY-2 and VBI-0, and of A. thaliana, ecotype Landsberg erecta (LE) cell line. Notably, we present an improved protocol for BY-2 transformation and cloning and extend the available plant cell lines methodology toward high-throughput technologies like fluorescent-based cell sorting and transcriptomics.