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Protein translation is tightly and precisely controlled by multiple mechanisms including upstream open reading frames (uORFs), but the origins of uORFs and their role in maize are largely unexplored. In this study, an active transposition event was identified during the propagation of maize inbred line B73. The transposon, which was named BTA for ‘B73 active transposable element hAT’, creates a novel dosage-dependent hypomorphic allele of the hexose transporter gene ZmSWEET4c through insertion within the coding sequence in the first exon, and results in reduced kernel size. The BTA insertion does not affect transcript abundance but reduces protein abundance of ZmSWEET4c, probably through the introduction of a uORF. Furthermore, the introduction of BTA sequence in the exon of other genes can regulate translation efficiency without affecting their mRNA levels. A transposon capture assay revealed 79 novel insertions for BTA and BTA-like elements. These insertion sites have typical euchromatin features, including low levels of DNA methylation and high levels of H3K27ac. A putative autonomous element that mobilizes BTA and BTA-like elements was identified. Together, our results suggest a transposon-based origin of uORFs and document a new role for transposable elements to influence protein abundance and phenotypic diversity by affecting the translation rate.

Many plant species exhibit genetic variation for coping with environmental stress. However, there are still limited approaches to effectively uncover the genomic region that regulates distinct responsive patterns of the gene across multiple varieties within the same species under abiotic stress.

By analyzing the transcriptomes of more than 100 maize inbreds, we reveal manycis- andtrans-acting eQTLs that influence the expression response to heat stress. Thecis-acting eQTLs in response to heat stress are identified in genes with differential responses to heat stress between genotypes as well as genes that are only expressed under heat stress. Thecis-acting variants for heat stress-responsive expression likely result from distinct promoter activities, and the differential heat responses of the alleles are confirmed for selected genes using transient expression assays. Global footprinting of transcription factor binding is performed in control and heat stress conditions to document regions with heat-enriched transcription factor binding occupancies.

Footprints enriched near proximal regions of characterized heat-responsive genes in a large association panel can be utilized for prioritizing functional genomic regions that regulate genotype-specific responses under heat stress.

CRISPR–Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR–Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR–Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR–Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on bothmore »CRISPR–Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

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Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

Abstract Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.

Abstract Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43, and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. A more complete set of UMRs and ACRs can be identified when chromatin data are aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are 6- to 12-fold enriched within regions between genomes. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only ∼5% being polymorphic between genotypes. However, the majority (71%) of UMRs that are shared between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instabilitymore »is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.« less

Abstract Transposable elements (TEs) pervade most eukaryotic genomes. The repetitive nature of TEs complicates the analysis of their expression. Evaluation of the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress reveals no evidence for genome-wide activation of TEs; however, some specific TE families generate transcripts only in stress conditions. There is substantial variation for which TE families exhibit stress-responsive expression in the different genotypes. In order to understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. The stress-responsive activation of a TE family can often be attributed to a small number of elements in the family that contains regions lacking DNA methylation. Comparisons of the expression of TEs in different genotypes revealed both genetic and epigenetic variation. Many of the specific TEs that are activated in stress in one inbred are not present in the other inbred, explaining the lack of activation. Among the elements that are shared in both genomes but only expressed in one genotype, we found thatmore »many exhibit differences in DNA methylation such that the genotype without expression is fully methylated. This study provides insights into the regulation of expression of TEs in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression variation. The highly repetitive nature of many TEs complicates the analysis of their expression. Although most TEs are not expressed, some exhibits expression in certain tissues or conditions. We monitored the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress. While genome-wide activation of TEs did not occur, some TE families generated transcripts only in stress conditions with variation by genotype. To better understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. In most cases, stress-responsive activation of a TE family was attributed to a small number of elements in the family. The elements that contained small regions lacking DNA methylation regions showed enriched expression while fully methylated elements were rarely expressed in control or stress conditions. The cause of varied expression in the different genotypes was due to both genetic and epigenetic variation. Many specific TEs activated by stress in one inbred were not present in the other inbred. Among the elements shared in both genomes, full methylation inhibited expression in one of the genotypes. This study provides insights into the regulation of TE expression in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression.« less