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Cis-regulatory elements are specific DNA sequences that control gene expression in a spatiotemporal manner, and variation within these elements represents a major source of phenotypic diversity and evolutionary innovation. Nevertheless, how regulatory elements evolve and shape gene expression remains poorly understood, particularly in plants. The well-resolved phylogeny of allopolyploid peanut (Arachis hypogaea) and its diploid progenitors,Arachis duranensisandArachis ipaensis, provides an ideal system to investigate the regulatory evolution at a lineage-specific level. By integrating comparative analyses of sequence similarity, chromatin accessibility, histone modifications, conserved noncoding sequences (CNSs), and gene expression, we reconstructed the evolutionary trajectories of Accessible Chromatin Regions (ACRs), where regulatory elements typically reside, and revealed their distinct contributions to homoeolog expression bias, unequal expressions between homoeologs. Most ACRs exhibited high sequence similarity, comparable chromatin accessibility, and conserved states for H3K4me3, H3K56ac, and H3K36me3, indicating regulatory stability after hybridization and polyploidization. However, a subset of novel ACRs emerged de novo from previously nonregulatory regions or through sequence mutations in preexisting ACRs, arising at different rates and evolutionary stages. Notably, even highly sequence-conserved ACRs exhibited substantial variation in chromatin accessibility, consistent with CNS composition differences and minor sequence variation, although causal relationships remain to be demonstrated. Our analyses further revealed a complex spectrum of CNS dynamics within the diploid-polyploid framework. Overall, our study provides empirical insights into the fine-scale evolution of plant regulatory landscapes and complements previous large-scale comparisons across distant lineages. 

Spatial transcriptomics is a disruptive technology that enables the identification of cell-type-specific transcripts en masse. However, the use of spatial transcriptomics for plant tissues has been challenging due to issues related to tissue preparation. Here, we present a protocol for preparing fresh frozen soybean tissues for spatial transcriptomics. We describe the steps for embedding, cryosectioning, fixation, staining, and imaging of soybean tissues.We then detail procedures for library preparation and sequencing. 

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. 

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 Cis-regulatory elements (CREs) are essential for regulating gene expression, yet their evolutionary dynamics in plants remain elusive. Here we constructed a single-cell chromatin accessibility atlas forOryza sativafrom 103,911 nuclei representing 126 cell states across nine organs. Comparative genomics betweenO. sativaand 57,552 nuclei from four additional grass species (Zea mays,Sorghum bicolor,Panicum miliaceumandUrochloa fusca) revealed that chromatin accessibility conservation varies with cell-type specificity. Epidermal accessible chromatin regions in the leaf were less conserved compared to other cell types, indicating accelerated regulatory evolution in the L1-derived epidermal layer ofO. sativarelative to other species. Conserved accessible chromatin regions overlapping the repressive histone modification H3K27me3 were identified as potentially silencer-like CREs, as deleting these regions led to up-regulation of gene expression. This study provides a comprehensive epigenomic resource for the rice community, demonstrating the utility of a comparative genomics approach that highlights the dynamics of plant cell-type-specific CRE evolution.