Maize Genomics and Genetics 2025, Vol.16, No.4, 182-201 http://cropscipublisher.com/index.php/mgg 198 Knowing which enhancers are naturally active in seeds (and under what conditions) helps choose candidates for enhancement. For instance, we identified an enhancer upstream of a key starch synthesis gene that is moderately active in mid-endosperm. If the goal is to increase starch content, one strategy could be to engineer that enhancer to have additional TF binding sites (e.g., more copies of O2 binding site) to drive higher expression of the starch gene. Since we see that enhancer is accessible, adding binding motifs for endosperm-active TFs (like O2, PBF) might boost its activity. Conversely, to reduce the expression of a gene (perhaps to redirect metabolic flow), one could introduce small mutations in its enhancer motifs to weaken TF binding (effectively what some natural SNPs do). Because our data pinpoint the exact motifs used by the plant (footprints), genome editing can be extremely precise - altering one or two base pairs in a motif can disrupt a TF’s binding. This is much cleaner than traditional methods (like RNAi or random mutagenesis) because it leaves the rest of the genome untouched and fine-tunes only the target gene’s regulatory control. We also emphasize that open chromatin regions are potential sites where epigenetic modifications could be applied. For example, technologies are emerging to deposit DNA methylation or repressive marks at specific sequences to downregulate gene activity (epigenome editing). The accessible regions we identified would be logical targets for such epigenetic editing, because adding a repressive mark to an enhancer or promoter that is normally open could shut down the associated gene. This could be reversible and potentially heritable, offering a new breeding tool that doesn’t alter the DNA code but the chromatin state. In crops, one might imagine using epigenome editing to temporarily silence a gene that limits yield, and our map tells us where to target such modifications (e.g., the promoter of a negative regulator of grain filling). Editing noncoding regulatory sequences can reproduce quantitative trait variation. Hendelman et al. (2021) in tomato, for instance, dissected a homeobox gene’s enhancer and created alleles that mimic natural diversity in fruit size. Similarly, in maize, a study by Wang et al. (2021) edited a promoter in a stem cell regulatory circuit to demonstrate how cis variation influences meristem architecture. We contribute to this growing evidence by showing an example in maize seed (NKD1) and by highlighting numerous new candidates in seed development that could be edited for improvement. The union of ATAC-seq and CRISPR thus holds great promise: ATAC-seq flags the critical regulatory nodes, and CRISPR allows us to modulate them in targeted ways. In practical breeding programs, this approach could be used to produce elite alleles faster. If a favorable allele is known (through GWAS) but is in a linkage drag context, one could recreate it by editing the native variety’s regulatory sequence as we did. For traits like seed size, composition, or stress tolerance, where often the differences are due to promoter or enhancer variants, genome editing guided by our open chromatin map could accelerate the incorporation of those traits. Another advantage is reducing pleiotropy: by tweaking a gene’s expression rather than knocking it out, one might avoid undesirable effects in other tissues or stages. For instance, completely knocking out NKD1 or ABI3 causes severe defects (nkd leads to defective aleurone, abi3 mutants cause precocious germination), but a slight promoter variant might improve one aspect (leaf growth or stress tolerance) without fully losing function. Indeed, our NKD1pro edited lines were perfectly viable and just had subtly longer leaves, implying the core seed functions of NKD1 remained intact. 7 Discussion and Outlook The results of this study indicate that chromatin accessibility does not merely follow genetic activity - it plays a crucial role in shaping seed traits. By turning on or off certain DNA regions at the right time, it controls when and where genes are activated. These changes will affect important traits, such as the size, strength of the seeds and the storage capacity of nutrients. For instance, at the beginning of the grain filling period, we found that the promoter of the starch production gene became more open. This openness enables these genes to be activated and drive the accumulation of starch, thereby controlling the carbohydrate content that eventually enters the seed. If the chromatin around these genes remains closed, the genes will not be activated even with the correct signals. Therefore, chromatin opening is a necessary condition for high starch production. We also found that the formation of a single aleurone layer in the endosperm requires the activation of NKD1 at the correct time. This occurs when its regulatory DNA becomes more open. If the chromatin does not undergo such changes due to
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