Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 190 significantly after 8 days. Previous studies have shown that it controls many genes related to seed filling. We found that the promoters of these target genes are open at this time. Many of them contain RY elements, which are known to be bound by ABI3/VP1. This supports the view that ABI19 plays an important role at this stage. Some NAC transcription factors, such as NAC128 and NAC130, also become active. They help manage starch-related genes. We found their binding sites in the open chromatin near starch genes, such as the CACG motif. These NACs are active when starch begins to accumulate, indicating their significant role at this stage. In the later stage of seed development (about 10 days to maturity) : After the seeds approach the dormant period, the level of abscisic acid (ABA) begins to rise. Meanwhile, many genes related to stress are activated. A key gene at this stage is Viviparous1 (VP1), which is the corn version of ABI3. VP1 is combined with RY components. This helps activate the genes needed in the later stage of embryo development. It can also inhibit genes that may start to germinate prematurely. At this stage, we observed a large number of RY motifs in the open chromatin regions. VP1 was also strongly expressed in our RNA sequencing results. This indicates that VP1 plays an important role as a transcription factor in the later stage of the seed. Other transcription factors also began to play a role, including heat shock factor (HSF) and the bZIP protein similar to ABI5. They help seeds cope with dryness. We found that heat shock elements (AGAAnnTTCT) were present in large quantities in the open areas at this stage. This might imply that the HSF is combined there. Genes such as ZmHSFA2 and ABI5 also showed higher expression. These genes may help activate the LEA gene and other protective genes. Chromatin also seems to undergo changes to support dormancy. Transcription factors like ZmABI4 (belonging to the AP2 family) may contribute to this. They can bind to certain open sites and shut down metabolic genes related to germination. These genes remain silent until the seeds absorb water. In addition to these broad categories, our analysis unearthed TF candidates for more specialized roles or cell-type specific roles. For example, the DOF family TF PBF (P-prolamin-box binding factor), which partners with O2 to regulate zein genes, likely binds accessible sites in late endosperm (we saw its AAAG motif footprints on zein gene promoters). PBF expression is indeed specific to endosperm and peaks during storage phase. Similarly, we observed motifs for MYB and WRKY factors enriched in bundle sheath- or aleurone-specific OCRs (from dissected tissue ATAC-seq), which suggests roles for those TFs in those specific seed tissues. For instance, WRKY TFs may be involved in the aleurone layer’s defense gene expression (Yang et al., 2016). To systematically pinpoint key regulators, we compiled a list of TF genes whose expression profiles matched the pattern of any OCR cluster and whose motif was enriched in those cluster’s sequences. This highlighted several candidates as “core” stage-specific TFs (some mentioned above). Many of these have literature support (like O2, ABI3, LEC2), lending credence to the ones that are more novel. For example, we identified a TF of the GRAS family (potentially ZmGRAS11) expressed during endosperm development; GRAS11 is reported to work downstream of O2 in promoting cell expansion, and we indeed saw its motif in mid-stage OCRs. Another novel one was a B3-domain factor apart from ABI3/VP1 – possibly ZmABI19 or ZmLEC1 (though LEC1 is a CCAAT-binding factor, it could show indirectly as a motif). These analyses underscore a temporal division of labor among transcription factors: MADS and ARFs early, various bZIPs and NACs in mid, and B3/ABI3 plus stress TFs late, all orchestrating development in sequence. 4.3 Regulatory network construction and core nodes One sub-network we constructed focuses on early embryogenesis and endosperm initiation. In this network, a central node is an SVP/AGL MADS-box TF (call it ZmMADSx) which appears to regulate a suite of early genes by binding their promoters (CArG motifs identified in those promoters’ OCRs). Those target genes include ones encoding hormone biosynthesis enzymes (e.g. YUCCA for auxin, a GA 20-oxidase for gibberellin) and other TFs like LEC2. Meanwhile, LEC2 itself forms another node that feeds into a slightly later set of targets – we placed LEC2 as regulating genes for seed storage initiation (like LEA precursors) and also upregulating ABI3/VP1 (as suggested by RY motifs upstream of Vp1). Thus, LEC2 is a predicted upstream activator of the maturation network. On the endosperm side, our early network includes NKD1/2 acting on aleurone differentiation genes (e.g. mrp1, a transfer layer regulator, and various Betl genes encoding transfer cell proteins). These interactions were
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