Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 192 auxin plays a crucial role in initiating endosperm growth immediately after fertilization. We discovered open chromatin regions near genes such as TAR1 (involved in auxin synthesis) and YUC (also involved in the same pathway). These open areas suggest that auxin related genes may be activated with the help of nearby accessible DNA in the early stage of seed development. Similarly, in the later stages of seed development, OCR becomes more accessible near abscisic acid (ABA) signal-related genes, including those encoding PP2C phosphatase and bZIP transcription factors such as ABI5. These changes are consistent with the role of ABA in seed dormancy preparation and the completion of development. In our KEGG analysis, "plant hormone signal transduction" was significantly enriched in genes related to OCR, which supports the view that hormone changes match chromatin openness changes during seed formation. Another broad category that emerged from enrichment analysis is gene regulation and chromatin organization itself. Several transcription factor activity GO terms (e.g. “sequence-specific DNA binding”) and chromatin modification terms (like “histone acetylation”) were enriched, meaning that many OCR-linked genes encode regulatory proteins. This includes the key transcription factors we identified (LEC1, O2, VP1, etc.), as well as components of chromatin remodeling complexes. For instance, genes encoding certain SNF2-family chromatin remodelers and chromatin assembly factors became accessible and expressed in specific stages, perhaps to facilitate the large-scale chromatin changes seeds undergo (like endoreduplication in endosperm cells or chromatin desiccation tolerance). Thus, the regulatory architecture appears to be somewhat self-reinforcing – changes in chromatin openness enable the expression of chromatin regulators that further modulate chromatin states downstream (Xie et al., 2023). 5.2 Enrichment of key developmental processes Focusing on specific developmental processes of interest – such as starch accumulation, cell differentiation, and hormone responses (as mentioned in the outline) – we find clear evidence that these processes are underpinned by coordinated chromatin accessibility changes. Maize seeds (notably the endosperm) accumulate large amounts of starch, and this process is tightly controlled at the transcriptional level by a suite of enzymes and regulators. Our ATAC-seq data show that many genes in the starch biosynthetic pathway become accessible during the mid-to-late endosperm development. For instance, Sh2 and Bt2, which encode the two subunits of ADP-glucose pyrophosphorylase (the rate-limiting enzyme in starch synthesis), had low chromatin accessibility in early development but showed strong promoter ATAC-seq peaks by 8-10 DAP, concurrent with their increased expression. Similarly, Sugary1 (a starch debranching enzyme) and Waxy (granule-bound starch synthase) genes also gained accessible promoters in the filling stage. GO enrichment already indicated starch biosynthesis was prominent; drilling down, we saw that specific transcription factors known to regulate starch-related genes also had changes in accessibility. ZmNAC128 and ZmNAC130, two NAC family TFs that positively regulate starch accumulation by activating starch synthase genes, showed increased expression in mid-development and had accessible promoter regions, presumably facilitating their own upregulation. It has been demonstrated that mutation in these NAC genes reduces starch content, highlighting their importance. In our data, OCRs containing NAC-binding motifs were found near starch synthase genes, suggesting NACs bind there to boost transcription. In summary, the process of starch accumulation is enriched in the sense that its key players (both enzymes and regulators) are among the genes with significant chromatin opening in the appropriate timeframe. During seed development, a burst of cell differentiation establishes the various cell types of the embryo (shoot meristem, root meristem, cotyledon tissues) and endosperm (aleurone, starchy cells, transfer cells, etc.). Our findings indicate that genes instrumental in cell differentiation show distinctive chromatin accessibility changes. For example, genes like Leafy Cotyledon1 (Lec1) and Baby Boom, which are involved in embryonic cell fate and somatic embryogenesis, were accessible primarily in early embryogenesis. Lec1 encodes an HAP3 subunit of CCAAT-binding factor and is a master regulator for embryo identity; we found its promoter to be accessible in early embryo stages (consistent with it being active then). In the endosperm, genes that mark specific lineages
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