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The rate and spectrum of somatic mutations can diverge from that of germline mutations. This is because somatic tissues experience different mutagenic processes than germline tissues. Here, we use nanorate sequencing (NanoSeq) to identify somatic mutations in Arabidopsis shoots with high sensitivity. We report a somatic mutation rate of 3.6x10^-8 mutations/bp, ~4-5x the germline mutation rate. Somatic mutations displayed elevated signatures consistent with oxidative damage, UV damage, and transcription-coupled nucleotide excision repair. Both somatic and germline mutations were enriched in transposable elements and depleted in genes, but this depletion was greater in germline mutations. Somatic mutation rate correlated with proximity to the centromere, DNA methylation, chromatin accessibility, and gene/TE content, properties which were also largely true of germline mutations. We note DNA methylation and chromatin accessibility have different predicted effects on mutation rate for genic and non-genic regions; DNA methylation associates with a greater increase in mutation rate when in non-genic regions, and accessible chromatin associates with a lower mutation rate in non-genic regions but a higher mutation rate in genic regions. Together, these results characterize key differences and similarities in the genomic distribution of somatic and germline mutations. 

DNA methylation is important to maintain genome stability, but alterations in genome-wide methylation patterns can produce widespread genomic effects, which have the potential to facilitate rapid adaptation. We investigate DNA methylation evolution in Arabidopsis thaliana during its colonization of the drought-prone Cape Verde Islands (CVI). We identified three high impact changes in genes linking histone modification to DNA methylation that underlie variation in DNA methylation within CVI. Gene body methylation is reduced in CVI relative to the Moroccan outgroup due to a 2.7-kb deletion between two VARIANT IN METHYLATION genes (VIM2 and VIM4) that causes aberrant expression of the VIM2/4 homologs. Disruptions of CHROMOMETHYLASE 2 (CMT2) and a newly identified DNA methylation modulator, F-BOX PROTEIN 5 (FBX5), which we validated using CRISPR mutant analysis, contribute to DNA methylation of transposable elements (TEs) within CVI. Overall, our results reveal rapid methylome evolution driven largely by high impact variants in three genes. 

Spontaneous epimutations—stochastic changes in cytosine methylation—can persist across generations in plants and are thought to contribute to phenotypic variation. Although epimutations are increasingly studied for their potential long-term effects, it remains unclear why their accumulation varies across genotypes. Here, we tracked DNA methylation across ten generations in ~400 mutation accumulation lineages derived from ~70ArabidopsisLer × Cvi recombinant inbred lines. Treating epimutation rates as quantitative molecular traits, we mapped a major QTL to a Cvi-derived deletion nearVIM2andVIM4, two genes involved in CG methylation (mCG) maintenance. We show that this deletion rapidly reduces genome-wide methylation to a lower steady-state and compromises mCG maintenance fidelity across generations, resulting in a ~1.5-fold increase in epimutation rates. Genotypes with elevated rates exhibited accelerated epigenetic drift and phenotypic divergence. Our findings support a punctuated-equilibrium model of mCG evolution, in which sudden disruptions to methylation homeostasis can destabilize epigenetic inheritance over longer time-scales. 

Gene expression and complex phenotypes are determined by the activity of cis-regulatory elements. However, an understanding of how extant genetic variants affect cis regulation remains limited. Here, we investigated the consequences of cis-regulatory diversity using single-cell genomics of more than 0.7 million nuclei across 172Zea mays(maize) inbreds. Our analyses pinpointed cis-regulatory elements distinct to domesticated maize and revealed how historical transposon activity has shaped the cis-regulatory landscape. Leveraging population genetics principles, we fine-mapped about 22,000 chromatin accessibility–associated genetic variants with widespread cell type–specific effects. Variants in TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR–binding sites were the most prevalent determinants of chromatin accessibility. Finally, integrating chromatin accessibility–associated variants, organismal trait variation, and population differentiation revealed how local adaptation has rewired regulatory networks in unique cellular contexts to alter maize flowering. 

While considerable knowledge exists about the enzymes pivotal for C₄photosynthesis, much less is known about thecis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C₄enzymes for five different grass species. This study spans four C₄species, covering three distinct photosynthetic subtypes:Zea maysandSorghum bicolor(NADP-dependent malic enzyme),Panicum miliaceum(NAD-dependent malic enzyme),Urochloa fusca(phosphoenolpyruvate carboxykinase), along with the C₃outgroupOryza sativa. We studied thecis-regulatory landscape of enzymes essential across all C₄species and those unique to C₄subtypes, measuring cell-type-specific biases for C₄enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C₄evolution. Besides promoter proximal ACRs, we found that, on average, C₄genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C₄evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution ofcis-regulation at these C₄loci. This study illuminates the dynamic and complex nature ofcis-regulatory elements evolution in C₄photosynthesis, particularly highlighting the intricatecis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C₃crop performance under changing climatic conditions. 

Heterochromatin is critical for maintaining genome stability, especially in flowering plants, where it relies on a feedback loop involving the H3K9 methyltransferase, KRYPTONITE (KYP), and the DNA methyltransferase CHROMOMETHYLASE3 (CMT3). The H3K9 demethylase INCREASED IN BONSAI METHYLATION 1 (IBM1) counteracts the detrimental consequences of KYP-CMT3 activity in transcribed genes.IBM1expression inArabidopsisis uniquely regulated by methylation of the 7th intron, allowing it to monitor global H3K9me2 levels. We show the methylated intron is prevalent across flowering plants and its underlying sequence exhibits dynamic evolution. We also find extensive genetic and expression variations inKYP,CMT3, andIBM1across flowering plants. We identifyArabidopsisaccessions resembling weakibm1mutants and Brassicaceae species with reducedIBM1expression or deletions. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity and loss of gene body DNA methylation, ascmt3mutants inA.thalianamitigate the deleterious effects of IBM1. 

Abstract Gene regulation in eukaryotes is partly shaped by the 3D organization of chromatin within the cell nucleus. Distal interactions between cis-regulatory elements and their target genes are widespread, and many causal loci underlying heritable agricultural traits have been mapped to distal non-coding elements. The biology underlying chromatin loop formation in plants is poorly understood. Dissecting the sequence features that mediate distal interactions is an important step toward identifying putative molecular mechanisms. Here, we trained GenomicLinks, a deep learning model, to identify DNA sequence features predictive of 3D chromatin interactions in maize. We found that the presence of binding motifs of specific transcription factor classes, especially bHLH, is predictive of chromatin interaction specificities. Using an in silico mutagenesis approach we show the removal of these motifs from loop anchors leads to reduced interaction probabilities. We were able to validate these predictions with single-cell co-accessibility data from different maize genotypes that harbor natural substitutions in these TF binding motifs. GenomicLinks is currently implemented as an open-source web tool, which should facilitate its wider use in the plant research community. 

SUMMARY WUSCHEL (WUS) is transcription factor vital for stem cell proliferation in plant meristems. In maize,ZmWUS1is expressed in the inflorescence meristem, including the central zone, the reservoir of stem cells.ZmWUS1overexpression in theBarren inflorescence3mutant leads to defects in inflorescence development. Here, single-cell ATAC-seq analysis shows thatZmWUS1overexpression alters chromatin accessibility throughout the central zone. The CAATAATGC motif, a known homeodomain recognition site, is predominantly observed in the regions with increased chromatin accessibility suggesting ZmWUS1 is an activator in the central zone. Regions with decreased chromatin accessibility feature various motifs and are adjacent toAUXIN RESPONSE FACTORgenes, revealing negative regulation of auxin signaling in the central zone. DAP-seq of ZmWUS1 identified the TGAATGAA motif, abundant in epidermal accessible chromatin compared to the central zone. These findings highlight ZmWUS1’s context-dependent mechanisms for stem cell maintenance in the inflorescence meristem.