Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 194 During the middle stage of seed growth (about 5 to 15 days), seeds mainly start to store nutrients. Manufacturing starch has become one of the most important tasks. The promoter regions of most of the key enzymes involved in starch production are open and strongly activated at this stage. In addition to starch, the synthesis of proteins has also begun to accelerate. Genes encoding ribosome parts, translation helper genes and storage proteins (such as zeolin) have become more active. Their promoter regions are also open, which is quite normal as the seeds are busy producing a large amount of protein. To keep all this going, the seeds need more energy. It promotes energy production by activating enzymes involved in glycolysis and the tricarboxylic acid cycle. For instance, the gene promoter of pyruvate kinase is in an open state and has a relatively high expression level. This helps ensure that the seeds have sufficient energy to maintain high levels of starch and protein production. Transcription factors like Opaque2 and O11 are active here, coordinating these storage processes within the endosperm. Meanwhile, cell division activity drops—mitosis-related genes lose accessibility as endosperm cells shift toward endoreduplication, and embryo development transitions from organ formation to growth and expansion. In the final phase (from ~20 DAP onward), metabolism slows down, while genes for stress protection, drying, and dormancy take over. We saw reduced chromatin openness at genes like starch synthases, which makes sense as starch accumulation finishes. At the same time, ABA-driven responses peak. LEA proteins, detoxification enzymes, and structural proteins linked to seed hardening show high expression and open promoters. Antioxidant systems, including peroxidases and the ascorbate-glutathione cycle, are turned on to handle reactive oxygen species during drying. We also saw signs of nutrient recycling, with chromatin opening in genes for proteases and lipases that might break down dying tissues or maternal support cells. From a control perspective, abscisic acid (ABA) and sugar signaling work together to help seeds enter a dormant state. Genes that help transport sugar usually contain a sugar response part (known as the SURE motif). Most of these genes remain off, possibly due to abscisic acid (ABA). Meanwhile, dormant genes of types like viviparous and DOG1 remain on and retain open DNA regions nearby. This shows how seeds utilize hormone and sugar signals to remain dormant and safe until they grow and mature. Visually, one can imagine a timeline of seed development where the “activity” of different pathways rises and falls in waves. At first, a developmental wave of growth and patterning (cell cycle, auxin) cresting early, followed by a wave of accumulation (starch/protein biosynthesis) cresting mid-development, and finally a wave of maturation (stress/dormancy) cresting late. Our chromatin accessibility profiles mirror these waves: early developmental pathways exhibit early OCR dynamics, mid pathways have mid-stage OCR peaks, and late pathways have late-stage OCR peaks. These dynamics are, in essence, the epigenomic signature of the maize seed’s developmental schedule. 6 Case study: Dynamic Chromatin Accessibility of Specific Genes at Key Stages 6.1 Chromatin accessibility changes at key endosperm development genes The endosperm of maize differentiates into multiple cell types, with the outer aleurone layer and inner starchy endosperm being the primary ones. Proper formation of the aleurone is essential for seed nutrient mobilization during germination and overall seed viability. A master regulator of aleurone cell fate in maize is the transcription factor Naked Endosperm 1 (NKD1), along with its duplicate NKD2. These are INDETERMINATE DOMAIN (IDD) family transcription factors required to specify aleurone versus starchy endosperm identity; nkd mutants develop extra layers of starchy cells in place of aleurone, hence the “naked endosperm” phenotype (Figure 2) (Hughes et al., 2023). We examined the ATAC-seq and RNA-seq profiles for the NKD1 gene and some of its known targets during seed development. Early in seed development (around 3–4 DAP), we found that the promoter region of NKD1 itself became highly accessible. This timing corresponds to endosperm cellularization and the onset of aleurone differentiation at the kernel periphery. The NKD1 gene’s promoter showed a clear ATAC-seq peak specifically in the peripheral
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