Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 191 drawn because NKD-binding IDD motifs (TTGTCG) were found in the promoters of Betl genes, and expression data shows Betl genes drop off in nkd mutants (Zhang et al., 2018). Moving to mid-development, the network we constructed shows Opaque11 (O11) as a hub in the endosperm regulatory network. O11 (a bHLH) is known to regulate other TFs such as Opaque2, PBF, and DOF3. Our data confirm this hierarchy: we saw O11’s binding E-box motif (CANNTG) in the accessible promoters of O2, PBF, and Dof3 during mid-stage, and those genes are indeed co-expressed. Thus in the network, O11 connects to O2, PBF, and DOF3, which in turn connect to their downstream targets (e.g. O2 and PBF jointly to zein storage protein genes, DOF3 and others to sugar metabolism genes). We also included ZmGRAS11 in this mid network, connecting it as a target of O2 (since O2 can transactivate ZmGRAS11) and as a regulator of endosperm cell expansion genes (perhaps cell wall biosynthesis genes). This is consistent with Feng et al. (2018) who identified interactions among these factors. Additionally, ABI19 appears in the mid network bridging embryo and endosperm: ABI19 likely receives input from LEC2 (RY motifs in ABI19 promoter) and outputs to both embryo regulators (like WOX genes for embryo patterning) and endosperm storage genes (it directly activates some late storage genes and coordinates with ABI3/VP1). We depicted ABI19 as a node linking to WOX2 and WUS (key embryo meristem genes), because our ATAC-seq showed ABI19 binding motifs on ZmWOX2 and ZmWUS2 promoters and luciferase assays confirmed ABI19 can bind those promoters. This suggests ABI19 helps set up the embryo body plan while also preparing the seed for maturation. For the late development network, ABI3/VP1 (the product of Vp1) is the central hub. In our network model, VP1 receives input from earlier factors LEC2 and ABI19 (since both likely help induce Vp1 expression), and then VP1 activates a broad suite of late genes. We connected VP1 to numerous LEA protein genes, late storage genes (e.g. late cysteine-rich peptides), and dormancy-related genes. We also have VP1 inhibiting some targets: known VP1/ABI3-repressed targets include germination-promoting genes like Amy1 (α-amylase) in cereal aleurone. We saw that the Amy1 promoter has RY motifs and remains inaccessible/low expressed until germination, implying VP1 bound and kept it closed (with the help of VAL repressors). Thus, in the network, VP1 links to Amy1 with a repression arrow. Another piece is the interplay with hormone signaling: VP1 works in concert with ABA. ABA can induce ABI5 (a bZIP) which then binds to late gene ABA-responsive elements. We included ABI5 in the network as being upregulated by VP1 and contributing to activating some late embryogenesis genes. As seeds dry, certain HSFs are also induced by ABA or stress, and our network shows an HSF node connecting to heat-shock protein and chaperone genes in late stage, reflecting their activation. 5 Functional Annotation and Developmental Pathways 5.1 GO and KEGG enrichment of OCR-associated genes We conducted gene ontology (GO) enrichment and KEGG pathway analysis on gene sets adjacent to open chromatin regions (OCRs), with a focus on genes located near OCRs and whose accessibility changed significantly at different stages of seed development. This helps us identify which biological processes may be affected by changes in chromatin openness. We found that many genes near OCR are associated with seed-related functions. These genes show a strong enrichment in some categories. For instance, GO terms such as "starch biosynthesis process" and "sucrose metabolism process" frequently appear in genes where chromatin accessibility increases in the middle and later stages of seed development. This is consistent with what we already know - the endosperm of corn produces a large amount of starch during this period. Important starch genes, such as Sh2, Bt2 and Wx, show strong ATAC-seq signals near their promoters or enhancers in the active state. These open areas may help promote the enrichment of starch-related GO terms. We also witnessed a similar situation in the KEGG analysis. The "starch and sucrose metabolism" pathway was significantly highly expressed in genes with later OCR opening (p < 0.001). This indicates that chromatin opening may be involved in activating these genes during the process of storage accumulation. We also found that many genes near the altered OCR are associated with GO terms related to plant hormones. These terms are mainly related to the synthesis and response of hormones. For instance, "auxin metabolic processes" and "responses to auxin" occur more frequently in genes near early OCR. This makes sense because
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