Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 185 when the seeds were still young. In contrast, late open OCRs were mostly close to genes related to nutrient storage and desiccation. These are common functions in mature seeds. Therefore, the timing and location of chromatin opening seem to match the activities of nearby genes at different stages. These time-based patterns weren’t just visible in the heatmap. We also ran a principal component analysis (PCA) on the ATAC-seq profiles, which clearly separated the samples along the first two axes. Developmental stages were spread out in order from earliest to latest along the first component, showing that most of the variation in chromatin accessibility comes from the seed's progression through time. From this, we could outline a sort of "pseudotime" map based on accessibility patterns that mirrors the actual developmental process of the seed. 3 Association between Open Chromatin and Gene Expression 3.1 Integration of OCR and transcriptome data By aligning ATAC-seq peaks with gene annotations and comparing to RNA-seq expression levels, we found a strong global correspondence between chromatin accessibility at gene promoters and the activation of those genes. In general, genes that were highly expressed in a given developmental stage had an accessible promoter in that stage, whereas genes that were transcriptionally silent tended to lack ATAC-seq peaks at their promoters. A positive correlation between promoter openness and gene expression was evident when plotting accessibility scores against mRNA levels for all genes: most points fell along an upward trend, indicating that increased chromatin accessibility is associated with higher transcription. For example, at 10 DAP (a stage of active endosperm filling), genes with the strongest ATAC-seq signals at their promoters (top decile of accessibility) showed on average a several-fold higher RNA-seq read count than genes with no detectable promoter accessibility. This broad correlation was also observed within gene clusters and pathways. Take the starch biosynthesis pathway: multiple enzymes in this pathway (such as ADP-glucose pyrophosphorylase, starch synthases, starch branching enzymes) are co-expressed during endosperm filling, and accordingly we detected open chromatin at the promoters of many of their genes specifically at the stages when starch is accumulating. One notable example is the Sh2 gene (encoding ADP-glucose pyrophosphorylase large subunit) – its promoter ATAC-seq signal is weak at early stages and becomes very strong at mid/late development, paralleling a jump in Sh2 transcript levels (consistent with Sh2’s known role in starch production in the endosperm) (Figure 1) (Yu et al., 2023). Such correspondence suggests that the establishment of open chromatin at these promoters is a key regulatory event enabling the transcription of storage product genes. To quantify the relationship, we computed Spearman’s correlation between promoter accessibility (ATAC-seq read count in the promoter region) and gene expression (RNA-seq read count) across our developmental series. The majority of genes exhibited positive correlations, and for a large subset the correlation was statistically significant (p < 0.01). These findings align with earlier reports in maize seedlings and other plants that accessible promoters generally mark actively transcribed genes. It is worth noting that while promoter accessibility and expression are broadly coupled, our analysis also revealed some exceptions: a small subset of genes had accessible promoters but low expression, or vice versa. Overall, our integrated analysis supports the intuitive model that during seed development, when a gene’s product is needed, its promoter becomes accessible (likely through chromatin remodeling events) and the gene is transcribed, whereas genes not needed remain in a closed chromatin state. This relationship provides confidence that ATAC-seq is capturing functionally relevant regulatory changes, and it allowed us to define sets of co-regulated genes based on combined chromatin–transcriptomic behavior. We didn’t just focus on promoters. We also looked at enhancers and the genes they might control during seed development. To find possible links, we used two main steps. First, we matched each open chromatin region (OCR) with the closest gene. Then, we compared how open the region was with how much the gene was expressed. Often, we saw that when an OCR became more open, the nearby gene became more active too. Many of these enhancers were found 5 to 100 kilobases away from the genes they might affect. These regions usually opened at certain times and matched changes in gene activity during development. For example, we found an OCR about 30 kb before a gene that helps make auxin. This region opened only during the early stages of seed growth. At the same time, the auxin gene showed high expression. This suggests the OCR
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