Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 195 endosperm tissue (when we analyzed dissected nucellus/aleurone vs. central endosperm), indicating that chromatin opening at NKD1 is localized to the future aleurone layer. By 6 DAP, NKD1 transcripts were abundant in the RNA-seq, and its promoter remained accessible, suggesting NKD1 is actively expressed through the differentiation phase. Notably, we identified an OCR not only at the core promoter but also in an intronic region of NKD1 that contains an ID1-binding motif; this could be an autoregulatory element or a site for feedback regulation by NKD itself or its dimerization partners. The accessibility of these NKD1 regulatory regions is consistent with the gene’s functional activation precisely when aleurone cells are being specified. Figure 2 NKD and SCR transcripts accumulate in the same spatial domain with levels determined by a feedback loop (Adopted from Hughes et al., 2023) Image caption: A) Quantitative RT-PCR of ZmSCR1 and ZmSCR2 transcripts in the Zmnkd1-Ds;Zmnkd2-Ds mutant, and ZmNKD1 and ZmNKD2 in the Zmscr1-m2;Zmscr1h-m2 mutant. Open circles are individual biological replicates, black crosses indicate the mean for each genotype. Statistical significance as calculated on log-transformed fold change data by Welch’s t-test (two-tailed) indicated above each plot: *P≤0.05; **P≤0.01; ***P≤0.001. B) In-situ hybridization to ZmNKD1 and ZmSCR1 in maize wild-type B73 apices. In each image the P2 primordium is outlined in blue, P3 in red, P4 in purple and P5 in green, as indicated in the adjacent cartoon diagram. Darker purple signal represents successful hybridization to each transcript of interest. Scale bars: 50μ m. C) Maximum likelihood phylogeny of the NKD genes in monocots. The NKD clade is highlighted in green, and the adjacent monocot clade in purple. Bootstrap values are displayed at each branch of the phylogeny (Adopted from Hughes et al., 2023)
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