Biology subjects

Slotkin, R. K.

Publications and source records attributed to Slotkin, R. K..

11 recordsLinked to original sources

bioRxiv2025

Elevated CO2 drives epigenetic reprogramming andchromatin dynamics in Arabidopsis thaliana

Atmospheric CO2 levels are rising rapidly, yet how plants respond at the genomic level remains poorly understood. The three-dimensional (3D) organization of chromatin in the nucleus is a key regulator of gene expression and environmental response, and changes in chromatin architecture can be transmitted across generations through epigenetic mechanisms. Here, we report that elevated CO2 is associated with broad 3D chromatin reorganization in Arabidopsis thaliana, spanning scales from chromosome-wide compartment identity to kilobase-resolution Local Chromatin Domains (LCDs) and LCD-Associated Loops (LALs). Using chromatin conformation capture and sequencing (Hi-C) across four mutant backgrounds, we show that these changes exhibit intergenerational persistence in the self-fertilized progeny of plants grown at elevated CO2, and that this persistence is sensitive to the Pol V branch of the RNA-directed DNA Methylation (RdDM) pathway rather than to upstream small RNA biogenesis. At kilobase resolution, we annotated 2,279 LCDs whose borders are enriched for transposable element loci occupied by the chromatin-silencing members Pol V and LHP1, and near a third of LCD borders are LALs. Elevated CO2 is associated with LAL attenuation and altered transcript abundance at LCD borders marked by H3K27me3, implicating coordinated RdDM and Polycomb group (PcG) activity in the chromatin response to elevated CO2. Integrated transcriptome and DNA methylation analysis across all five genetic backgrounds revealed widespread changes in gene and transposable element abundance. These findings support a model in which CO2-associated chromatin reorganization confers transcriptional plasticity through persistent chromatin states shaped by the RdDM and PcG pathways.

plant biology↗

bioRxiv2025

Somatic mobility of transposons is explosive and shaped by distinct integration biases in Arabidopsis thaliana

BackgroundThe movement of transposable elements (TEs) in somatic cells generates genetic and phenotypic diversity across the soma of individual organisms. This process is especially important in plants that display a sessile lifestyle, can regenerate from somatic cells, and continuously establish their germline from somatic stem cells. Yet, the scale and integration biases by which TEs may shape the plant soma remain unknown. ResultsWe adapt high-throughput TE sequencing and introduce a new computational pipeline, "deNOVOEnrich", to identify and analyse somatic transposition events in Arabidopsis thaliana. We detect [~]200,000 new TE insertions across wild-type and epigenetic mutant backgrounds, revealing en masse active transposition in the plant soma. Similar to germline dynamics, somatic integration is non-random and TE-specific. Focusing on the ONSEN, EVADE and AtCOPIA21 families, we find they preferentially integrate into chromosomal arms and genic DNA, and are enriched over H2A.Z, H3K27me3 and H3K4me1 labelled chromatin. However, ONSEN dynamically targets active and open chromatin, while EVADE displays a depletion in these regions, consistent with niche targeting at the local epigenetic level and spatial distribution across the genome ecosystem. We further demonstrate that environmentally-responsive genes such as resistance (R) genes and biosynthetic gene clusters are preferred integration targets, highlighting the adaptive potential of somatic transposition. ConclusionsOur work demonstrates that transposon amplification is an explosive and structured event that generates cellular mosaicism in the soma of A. thaliana. We speculate that this phenomenon is common across plants under permissive conditions, both in experimental and natural settings.

plant biology↗

bioRxiv2024

A transposable element insertion in IAA16 interrupts normal splicing and generates a novel dicamba resistance allele in Bassia scoparia

A dicamba-resistant population of kochia (Bassia scoparia) identified in Colorado, USA in 2012 was used to generate a synthetic mapping population that segregated for dicamba resistance. Linkage mapping associating dicamba injury with genotype derived from restriction-site-associated DNA sequencing identified a single locus in the kochia genome associated with resistance on chromosome 4. A mutant version of Auxin/Indole-3-Acetic Acid 16 (AUX/IAA16; a gene previously implicated in dicamba resistance in kochia) was found near the middle of this locus in resistant plants. Long read sequencing of dicamba-resistant plants identified a recently inserted Ty1/Copia retrotransposon near the beginning of the second exon of AUX/IAA16, leading to disruption of normal splicing. A molecular marker for this insertion allows for rapid detection of resistance. Stable transgenic lines of Arabidopsis thaliana ectopically expressing the mutant and wildtype alleles of AUX/IAA16 were developed. Arabidopsis thaliana plants expressing the mutant AUX/IAA16 allele grew shorter roots on control media. However, transgenic root growth was less inhibited on media containing either dicamba (5 M) or IAA (0.5 M) when compared to non-transgenic plants or those expressing the wildtype allele of AUX/IAA16. In vitro assays indicate reduced binding affinity and more rapid dissociation of the mutant AUX/IAA with TIR1 in the presence of several auxins, and protein modeling suggests the substitution of the glycine residue in the degron domain of AUX/IAA16 is especially important for resistance. A fitness cost associated with the mutant allele of AUX/IAA16 has implications for resistance evolution and management of kochia populations with this resistance mechanism. SignificanceAuxin mimics are amongst the most important herbicides in modern agriculture. Evolution of weeds that are resistant to these herbicides threatens sustainable crop production. Understanding the basis of auxin herbicide resistance informs the development of improved weed control technologies. Additionally, auxin-resistant mutations and their pleotropic effects help us understand auxin perception and signalling. We describe a transposable element insertion within an herbicide target site gene that alters splicing and reduces synthetic and natural auxin perception.

plant biology↗

bioRxiv2022

Plastid double-strand RNA transgenes trigger small RNA-based gene silencing of nuclear-encoded genes

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escape to the cytoplasm occurs. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment deficient plants. Furthermore, plastid-expressed double-stranded RNA (dsRNA) with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs broadly, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open new fields of study in plastid development, compartmentalization and small RNA biogenesis.

plant biology↗

bioRxiv2021

An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

plant biology↗

bioRxiv2020

A new role for histone demethylases in the maintenance of plant genome integrity

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development and adaptation to environmental fluctuations in most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the inheritance of epigenetic imprints, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases in plants contribute towards maintaining distinct transcriptional states during development and help safeguard genome integrity following sexual reproduction.

plant biology↗

bioRxiv2020

Long-Read cDNA Sequencing Enables a 'Gene-Like' Transcript Annotation of Arabidopsis Transposable Elements

High-quality transcript-based annotations of genes facilitates both genome-wide analyses and detailed single locus research. In contrast, transposable element (TE) annotations are rudimentary, consisting of only information on location and type of TE. When analyzing TEs, their repetitiveness and limited annotation prevents the ability to distinguish between potentially functional expressed elements and degraded copies. To improve genome-wide TE bioinformatics, we performed long-read Oxford Nanopore sequencing of cDNAs from Arabidopsis lines deficient in multiple layers of TE repression. We used these uniquely-mapping transcripts to identify the set of TEs able to generate mRNAs, and created a new transcript-based annotation of TEs that we have layered upon the existing high-quality community standard TAIR10 annotation. The improved annotation enables us to test specific standing hypotheses in the TE field. We demonstrate that inefficient TE splicing does not trigger small RNA production, and the cell more strongly targets DNA methylation to TEs that have the potential to make mRNAs. This work provides a transcript-based TE annotation for Arabidopsis, and serves as a blueprint to reduce the genomic complexity associated with repetitive TEs in any organism.

plant biology↗

bioRxiv2020

Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis

Environmentally induced changes in the epigenome help individuals to quickly adapt to fluctuations in the conditions of their habitats. Here we explored those changes in Arabidopsis thaliana plants subjected to multiple biotic and abiotic stresses, and identified transposable element (TE) activation in plants infested with the green peach aphid, Myzus persicae. We performed a genome-wide analysis of DNA methylation, mRNA expression, mRNA degradation and small RNA accumulation. Our results demonstrate that aphid feeding induces loss of methylation of hundreds of loci, mainly TEs. This loss of methylation has the potential to regulate gene expression and we found evidence that it is involved in the control of key plant immunity genes. Accordingly, we find that mutant plants deficient in epigenetic silencing show increased resistance to M.persicae infestation. Collectively, our results show that changes in DNA methylation play a significant role in the regulation of the plant transcriptional response and induction of defense response against aphid feeding.

plant biology↗

bioRxiv2019

MicroRNA function transitions from regulating developmental genes to transposable elements during the maturation of pollen

microRNAs play important roles to control the development of eukaryotic organisms. Both animal and plant microRNAs are essential for the spatio-temporal regulation of development but together with this role, plant microRNAs also control transposable elements and stimulate the production of epigenetically-active small interfering RNAs. This last role is evident in the plant male gamete containing structure, the male gametophyte or pollen grain, but how the dual role of plant microRNAs is integrated during its development is unknown. Here, we provide a detailed analysis of microRNA dynamics during pollen development and their genic and transposable element targets using small RNA and mRNA cleavage (PARE) high-throughput sequencing. Furthermore we uncover the microRNAs loaded in the two main Argonaute proteins in the mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a reprogramming from microRNAs focused on the control of development to microRNAs regulating transposable element control.

plant biology↗

bioRxiv2019

Arabidopsis RNA Polymerase IV generates 21-22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes

The plant-specific RNA Polymerase IV (Pol IV) transcribes heterochromatic regions, including many transposable elements, with the well-described role of generating 24 nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs target DNA methylation back to transposable elements to reinforce the boundary between heterochromatin and euchromatin. In the male gametophytic phase of the plant life cycle, pollen, Pol IV switches to generating primarily 21-22 nt siRNAs, but the biogenesis and function of these siRNAs has been enigmatic. In contrast to being pollen-specific, we identified that Pol IV generates these 21-22 nt siRNAs in sporophytic tissues, likely from the same transcripts that are processed into the more abundant 24 nt siRNAs. The 21-22 nt forms are specifically generated by the combined activities of DICER proteins DCL2/DCL4 and can participate in RNA-directed DNA methylation. These 21-22 nt siRNAs are also loaded into ARGONAUTE1, which is known to function in post-transcriptional regulation. Like other plant siRNAs and microRNAs incorporated into AGO1, we find a signature of genic mRNA cleavage at the predicted target site of these siRNAs, suggesting that Pol IV-generated 21-22 nt siRNAs may function to regulate gene transcript abundance. Our data provides support for the existing model that in pollen Pol IV functions in gene regulation.

plant biology↗

bioRxiv2019

Highly expressed maize pollen genes display coordinated expression with neighboring transposable elements and contribute to pollen fitness

In flowering plants, the haploid male gametophyte (pollen) is essential for sperm delivery, double fertilization, and subsequent initiation of seed development. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene regulation and function is not clearly understood. To identify components of these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and isolated sperm cells) via RNA-seq. We found that, in contrast to Arabidopsis TE expression in pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinated expression was observed between the most highly expressed protein-coding genes and neighboring TEs, specifically in both mature pollen and sperm cells. To test the hypothesis that such elevated expression correlates with functional relevance, we measured the fitness cost (male-specific transmission defect) of GFP-tagged exon insertion mutations in over 50 genes highly expressed in pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in genes highly expressed only in seedling or primarily in sperm cells (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation. The gamete expressed2 (gex2) gene was the single sperm cell gene associated with reduced transmission when mutant (<35% for two independent insertions), and also triggered seed defects when crossed as a male, supporting a role for gex2 in double fertilization. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes.\n\nAuthor SummaryIn flowering plants, pollen is essential for delivering sperm cells to the egg and central cell for double fertilization, initiating the process of seed development. In plants with abundant pollen like maize, this process can be highly competitive. In an added layer of complexity, growing evidence indicates expression of transposable elements (TEs) is more dynamic in pollen than in other plant tissues. How these elements impact pollen function and gene regulation is not well understood. We used transcriptional profiling to generate a framework for both detailed analysis of TE expression and quantitative assessment of gene function during maize pollen development. TEs are expressed early and persist, many showing coordinate activation with highly-expressed neighboring genes in the pollen vegetative cell and sperm cells. Measuring fitness costs for a set of over 50 mutations indicates a correlation between elevated transcript level and gene function in the vegetative cell. Finally, we establish a role in fertilization for the gamete expressed2 (gex2) gene, identified based on its specific expression in sperm cells. These results highlight maize pollen as a powerful model for investigating the developmental interplay of TEs and genes, as well as for measuring fitness contributions of specific genes.

plant biology↗