Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 183 more exposed and not wrapped tightly by nucleosomes, often overlaps with key DNA control sites known as cis-regulatory elements (CREs). These include promoters and enhancers that serve as landing pads for TFs and other chromatin-related proteins. In plants, large-scale mapping has shown that many of these open areas are located near gene promoters, and the more open a promoter is, the more likely that gene is being used. A good example is ATAC-seq, a method that detects open DNA. Studies using ATAC-seq in plants reveal that about 15%-30% of open regions sit close to transcription start sites. Genes near these regions tend to be turned on, while those far away often need enhancer regions—parts of the genome that can loop over long distances to reach and affect a gene (Lu et al., 2016; Zhang et al., 2022). Chromatin accessibility is an important epigenetic feature that regulates gene expression during plant development. Although transcriptional changes during maize seed development have been extensively studied, the dynamic changes in chromatin accessibility have not been deeply analyzed. In this study, we systematically analyzed the genome-wide open chromatin regions (OCRs) in multiple developmental stages of maize seeds using ATAC-seq (assay for transposase-accessible chromatin sequencing) technology, and combined RNA-seq transcriptome data to reveal the dynamic changes in chromatin accessibility with developmental stages, which were highly correlated with gene expression activity. We found that OCRs were mainly enriched in promoter and enhancer regions, and motif analysis further identified multiple transcription factor families such as bZIP, MYB, and NAC that may be involved in developmental regulation. Functional annotation indicated that OCR-related genes were significantly enriched in key biological processes such as starch synthesis, hormone signaling, and embryo development. This study has drawn a panoramic map of chromatin accessibility during maize seed development, revealing potential regulatory elements and key regulatory factors, providing a theoretical basis and target resources for maize epigenetic research and molecular breeding. 2 Chromatin Accessibility Changes during Seed Developmental Stages 2.1 Temporal changes in OCR number and abundance Maize seeds possess thousands of genomic regions that become accessible (or inaccessible) as development proceeds. We identified on the order of 40 000-60 000 high-confidence OCRs at each developmental stage, indicating that a substantial portion of the maize genome is involved in regulatory activity during seed formation. In the very early stages immediately post-fertilization, when the endosperm is still a coenocyte and the embryo is just beginning to divide, we detected the fewest OCRs (roughly 3.8×104). As development advanced into the cellularization of the endosperm and differentiation of embryonic organs, the number of OCRs increased markedly – peaking at nearly 6.0×104 open regions by the middle of the seed-filling phase. This trend suggests an overall opening of the chromatin landscape as the seed’s cellular complexity and metabolic activity ramp up. The expansion in OCR count may reflect the activation of many tissue-specific genes and cis-elements required for storage product biosynthesis, cell expansion, and other processes that dominate mid-development. After this peak, we observed that the total number of OCRs plateaued or even modestly declined in later stages of maturation (e.g. approaching desiccation), hinting that chromatin accessibility might contract again as seed development concludes. A similar pattern of initial increase and later reduction in accessible sites has been reported in other plant developmental contexts, such as maize leaf maturation and inflorescence development. 2.2 Genomic distribution of accessibility changes (promoters, enhancers, intergenic regions) We next examined where in the genome these OCRs are located and how their genomic distribution changes with development. Consistent with prior studies in plants, we found that promoter regions (defined here as a few hundred base pairs upstream of transcription start sites) are highly enriched for open chromatin. Across stages, roughly 20%–25% of all OCRs were located in promoters or genic upstream regions. These promoter-proximal OCRs include sites like core promoters and 5′ UTR regulatory sequences that are accessible when the associated gene is active. We observed that many genes which are broadly expressed during seed development (e.g. genes for core metabolism or cell maintenance) have constitutively accessible promoters at all stages, whereas genes with stage-specific expression often showed promoter accessibility only at the corresponding stage. For instance, the promoter of a gene encoding a starch synthase enzyme was open (ATAC-seq peak present) during the mid-to-late endosperm development when starch was actively being synthesized, but not accessible at the earliest embryonic
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