Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 196 We next looked at NKD1 target genes. One direct target is the gene MRP1 (Myb-related protein-1), a transcription factor that NKD1 activates to promote transfer cell (basal endosperm transfer layer, BETL) differentiation. In our data, MRP1 showed a distinct open chromatin profile: its promoter was accessible slightly later than NKD1 – around 4–6 DAP – aligning with the formation of the BETL after the aleurone begins to differentiate. MRP1 expression and accessibility were localized to the basal endosperm region, as expected. Interestingly, MRP1’s promoter has multiple IDD (NKD-binding) motifs, and we saw footprints on those motifs in the ATAC-seq data from basal endosperm nuclei, strongly suggesting NKD1/2 proteins bind there to activate MRP1. Furthermore, genes that MRP1 in turn regulates (the so-called BETL genes encoding transport proteins like BETL1, BETL2, etc.) also showed stage- and tissue-specific chromatin accessibility: their promoters opened and transcripts accumulated in the basal endosperm slightly after MRP1 was expressed. For example, the promoter of Betl1 (a defensin-like transport protein) had no ATAC signal at 3 DAP, became accessible at 6 DAP specifically in basal endosperm tissue, and was highly accessible by 8 DAP – matching the Betl1 expression pattern. This cascade – NKD1 accessible first, then MRP1, then BETL genes – paints a coherent picture of the gene regulatory cascade in aleurone/BETL differentiation, each step evident in our chromatin data. One of the important functions of NKD1 is to ensure that only one layer of batter is formed on the outside. NKD1 and NKD2 achieve this by inhibiting a gene called Thick aleurone layer 1 (Thk1). If Thk1 remains active, it may result in more than one layer of paste powder. We examined the area around the Thk1 gene. In seeds that simultaneously lack both NKD1 and NKD2, the chromatin near Thk1 is in an open state. This result is consistent with the research findings of Gontarek et al. (2016). However, in normal seeds with NKD1 present, the same area remains closed. This indicates that NKD1 may introduce other proteins to help Thk1 remain closed. It may also block the gene in a roundabout way. We also discovered a possible silent area upstream of Thk1. This region contains IDD motifs and is more closed in normal seeds than in mutants. However, we lack a large amount of mutant data, so this part of the research still needs further study. Despite this, NKD1 may act in synergy with repressor proteins at certain sites. This is in line with another point we saw before - open chromatin does not always mean that genes are activated. Sometimes, it allows repressor proteins to enter and shut down genes. In short, the case of NKD1 shows that observing the openness of chromatin can explain how seed tissues such as Aladdin and BETL are formed. When NKD1 is activated, it triggers a chain reaction of gene activity. We can achieve this by tracking the time when the open region of each gene locus appears. 6.2 Candidate regulatory elements linked to seed maturation traits One powerful application of our chromatin accessibility map is the identification of regulatory DNA associated with important seed traits. Many agronomic traits, such as seed size, composition (starch/protein/oil content), and stress tolerance of seeds, are quantitative and controlled by multiple loci. Genome-wide association studies (GWAS) in maize have identified numerous trait-associated single nucleotide polymorphisms (SNPs) that often lie in noncoding regions. A recurring challenge is to pinpoint which SNPs affect gene regulation and how. Our ATAC-seq data help address this by showing which noncoding regions are likely functional (being open chromatin) and therefore which trait-associated SNPs might reside in bona fide regulatory elements. As part of this study, we cross-referenced a list of known seed trait QTL and GWAS hits with our OCR locations. We found that a substantial proportion of trait-associated SNPs for seed traits are indeed located within or very close to OCRs in our dataset (far more than would be expected by chance, which is consistent with other findings that phenotype-associated variants often overlap accessible chromatin). For example, a known QTL for kernel weight maps near the gene ZmABI19. Our data show an OCR (enhancer) in the 5′ region of ZmABI19 that is highly accessible in mid-development. Interestingly, within this enhancer resides a SNP that had been associated with variation in kernel size in a diversity panel. The SNP allele correlated with larger kernels is predicted to create a stronger binding site for an ABI3/VP1 protein (changing the sequence closer to the RY consensus). The chromatin at ZmABI19’s enhancer was accessible regardless of SNP variant, but the downstream effect might be that one allele of this OCR drives higher ABI19 expression. This illustrates how an open chromatin region harboring a nucleotide polymorphism can contribute to phenotypic diversity – a stronger enhancer could lead to
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