Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 186 might help start auxin production, which is important early on. We also saw OCRs near LEA protein genes open later in development. This matched the increase in LEA gene expression during those stages. While this doesn’t prove the enhancer-gene link directly, the pattern fits what we know about plant development. It also agrees with recent 3D genome studies in maize. Those studies showed that chromatin loops can bring faraway regulatory regions close to the genes they control, especially when the genes are active (Zhou et al., 2024a). Figure 1 The bimolecular fluorescence complementation between different mutants of Bt2 and Sh2. Fluorescence signals indicate that the Bt2 protein retains its interaction with the large subunit Sh2 (Adopted from Yu et al., 2023) 3.2 Co-regulated gene clusters and functional enrichment Once we saw that open chromatin often matches gene activity, we wanted to look closer. We tried to find sets of genes that changed in both openness and expression at the same time. We also wanted to learn what these genes do. So, we grouped them by looking at two things: how their promoter regions opened or closed over time, and how their expression levels went up or down during those same stages. This gave us a few clear gene groups that seemed to work together. For instance, one group showed open promoters and high expression early, between 0 and 3 DAP, but then slowed down later. Another group stayed quiet in the beginning but became active and open from about 8 to 15 DAP. Some genes stayed on the whole time, and others only turned on briefly. After making these clusters, we used GO term and KEGG pathway tools to check what each group might be doing. These tools helped us guess the key role of each gene set. Some were tied to early growth, while others helped with storage or stress later on. In short, grouping by openness and activity gave us a good idea of what different genes are doing at each stage. Genes in the “early-stage” cluster (high accessibility/expression in the young seed) were significantly enriched for GO categories related to DNA replication, cell cycle, chromatin organization, and embryonic pattern formation. This makes sense because shortly after fertilization, the endosperm undergoes rapid nuclear divisions and the embryo starts forming fundamental structures. Indeed, early-stage accessible genes included those encoding core
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