Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 189 Another highly enriched motif was the RY-repeat (CATGCA[TG]), which is the hallmark binding site for B3 domain transcription factors of the ABI3/VAL family. We found RY motifs particularly enriched in OCRs associated with late embryogenesis genes and seed maturation genes. This aligns with the presence of B3 TFs like Viviparous1 (ZmABI3) and FUSCA3-like factors in seeds. ABI3/VP1 is known to bind RY elements to activate late maturation genes (like LEA protein genes) and also to maintain repression of germination genes during seed development. The presence of accessible RY-containing elements in our late-stage ATAC data suggests that ABI3 and related B3 TFs are engaging those sites. Interestingly, VAL repressors also recognize RY motifs, so the context (cofactor presence, chromatin marks) likely determines whether a given RY element acts as an activation site or a repression site. Our data showed RY motifs in both promoter OCRs of late genes (likely activation by ABI3) and in some early accessible but silent sites (likely binding of VAL early, then replaced by ABI3 late) – supporting the dynamic use of this motif. Auxin-responsive elements (AuxREs) were another prominent category: the TGTCTC motif (and slight variants thereof) was enriched in OCRs during early embryo development. This sequence is the binding site for Auxin Response Factors (ARFs), which mediate auxin signaling. Auxin is a key signal at the start of seed development, and ARFs like ARF5/MONOPTEROS are crucial for embryonic patterning. The detection of AuxRE motifs in early-stage open regions aligns with the idea that auxin signaling pathways are transcriptionally active then. We also found that many of these AuxRE-containing OCRs were near genes involved in embryo axis formation and organ initiation. Supporting evidence comes from known ARF targets and regulators of auxin distribution (e.g. YUCCA auxin biosynthesis genes) which had accessible AuxRE motifs upstream when those genes were being expressed. Furthermore, using a motif clustering approach, we identified several ARF family members with potential activity: ARF25, ARF34, and ARF35 motifs (predicted by slight differences in base preferences) appeared in our embryo-specific OCR cluster. This is consistent with recent reports that specific ARFs have predominant roles in maize embryo morphogenesis. 4.2 Identification of stage-specific transcription factors The pattern of motif enrichment in accessible chromatin provides strong clues about which transcription factors are most active at each developmental stage. To distill this information, we cross-referenced the enriched motifs and footprints with the expression patterns of the corresponding TF genes. We found a remarkable concordance: transcription factor families whose motifs were enriched in early-stage OCRs tend to have members that are highly expressed in early seed development, and similarly for mid or late stages. This enabled us to propose a set of key TFs acting at specific times: Early seed development (0–3 DAP): Our data point to the involvement of MADS-box and auxin-related TFs. In particular, we identified a maize MADS-domain factor most similar to Arabidopsis AGL15/AGL18 that is highly expressed in the developing endosperm during cellularization, and whose binding sites (CArG motifs) are accessible at that time. This suggests it may play a role in endosperm differentiation. Another early regulator is auxin-responsive TFs: multiple ARF genes (e.g. ZmARF34, ZmARF35) show peak expression in the developing embryo around the transition from proembryo to organogenesis, and correspondingly, ARF motifs are prominent in embryo OCRs. Additionally, we saw evidence of the B3 TF LEC2 (LEAFY COTYLEDON2) or its maize ortholog being active early: the LEC2 motif (RY element) appears even in some early OCRs, and ZmLEC2 transcripts were detected in the 4–6 DAP embryo. LEC2 is known to initiate somatic embryogenesis and influence storage gene expression later, so its early presence might prime the embryo for maturation. Mid-stage seed development (4-8 days): At this stage, the embryo begins to form its basic organs. Meanwhile, the endosperm stops dividing and starts to store nutrients. We found that some bZIP transcription factors become active during this period. One key factor is Opaque2 (O2). It begins to appear in the maize endosperm after 6 to 8 days. O2 helps to activate genes encoding storage proteins. We also found that its binding site (TGACGT, a part of the P-box) is more common in open chromatin regions (OCRs) that become accessible after 6 to 10 days. Another important factor we discovered is ABI19. Like VP1 and ABI3, it belongs to the B3 domain group. ABI19 is involved in controlling the growth of the embryo and endosperm. Its expression level in both tissues increases
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