Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 187 cell-cycle regulators (e.g. cyclins, DNA polymerase subunits) and factors for morphogenesis (such as embryo sac development genes). Many of these early genes have to be tightly controlled once their window closes; accordingly, their promoters lost accessibility as development progressed and cell division slowed. In contrast, genes in the “late-stage” cluster (accessible/expressed primarily during seed filling and maturation) were enriched for processes like starch and sucrose metabolism, storage protein accumulation, abiotic stress response (desiccation tolerance), and hormone signaling. Key metabolic genes such as Sh2 and Bt2 (ADP-glucose pyrophosphorylase subunits for starch biosynthesis) and Zein family genes (storage proteins) fell into this category, consistent with the known timing of reserve deposition in endosperm. Their promoters became accessible in mid-development, likely under the control of endosperm master regulators (like O2 and PBF for zeins), and remained open through maturation to drive high-level expression. The late cluster was also rich in genes for LEA proteins and small heat-shock proteins (involved in dehydration tolerance), matching the seed’s preparatory steps for dormancy. GO terms for response to ABA and seed maturation were significantly overrepresented here, reflecting the ABA-mediated gene activation in late seed development. Another interesting cluster was one with biphasic or persistent expression (genes on at multiple stages). This set included many “housekeeping” genes (basic metabolism, translation, etc.) as well as some regulatory genes that act across stages. Their promoters were constitutively accessible, which is in line with their continuous requirement. GO analysis for this cluster showed enrichment in primary metabolic processes and protein synthesis, indicating these genes support general cellular functions throughout development. Notably, some of these genes still showed subtle changes in expression (e.g. upregulated during the intense growth phase), and correspondingly slight increases in ATAC signal, but overall they remained accessible at all times – a possible signature of genes that maintain the basal physiological state of the seed. We also connected our co-regulation findings to known gene regulatory networks. For example, we observed that many genes controlled by the Opaque11 network in the endosperm (as identified by Feng et al. (2018)) fell into a common cluster: they all start being expressed around the onset of endosperm cellularization and peak during storage accumulation. In our data, these genes (including several zein storage protein genes, pyruvate phosphate dikinase for carbon metabolism, etc.) indeed had promoters that became accessible around 4–6 DAP and remained open thereafter, consistent with O11’s timing of action. The enrichment analysis of that cluster highlighted terms like nutrient reservoir activity and transcription factor activity, illustrating that it contained both metabolic genes and regulatory genes co-activated in the mid-stage endosperm. Similarly, an early cluster contained both NKD1/2 and their downstream target genes (like Betl genes for basal endosperm transfer layer formation), all showing early accessibility and expression, which corresponds to NKD’s role in early endosperm patterning. 3.3 Transcriptional features of active vs. repressive OCRs While most open chromatin regions in our data were associated with gene activation, we did identify a subset of OCRs that did not correspond to actively expressed genes. These “accessible-but-silent” regions suggest that not all accessible chromatin is permissive for transcription-some may be bound by repressors or correspond to poised elements. For example, we found a number of gene promoters that were accessible (as indicated by ATAC-seq peaks) even when the gene’s mRNA was either low or undetected. Closer inspection revealed that many of these genes are ones that become active only under specific conditions or later in development (outside the sampled stages), or they are tissue-specific within the seed (e.g. restricted to a cell layer with low abundance in whole-seed RNA). However, some cases were puzzling – genes with accessible promoters and apparently no expression in our dataset. Interestingly, motif analysis on these accessible-but-inactive promoters showed enrichment of binding sites for known transcriptional repressors. One notable motif was the RY cis-element (CATGCA), which is recognized by B3-domain factors of the VAL (VP1/ABI3-Like) family that recruit Polycomb complexes to silence gene expression during certain stages. Indeed, studies in Arabidopsis have shown that VAL1/VAL2 bind to DNA and recruit PRC2 (Polycomb Repressive Complex 2) to establish H3K27me3 marks, keeping seed maturation genes off until the appropriate time. We observed that some late embryogenesis genes in maize (analogous to those VAL would target) had accessible chromatin at their promoters in early development but were not yet expressed – plausibly because repressive complexes were in place. As the seed transitions to maturation, these
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