Maize Genomics and Genetics 2025, Vol.16, No.6, 325-333 http://cropscipublisher.com/index.php/mgg 328 3.3 Coupling between genomic methylation patterns and transcriptional regulation How exactly are gene expression and DNA methylation related? There is actually no uniform answer to this question. Methylation in different regions and of different types also involves different ways. For instance, if CG methylation occurs in the genome, it is often associated with a "moderate" expression level. However, if it is concentrated on the promoter, especially at the CG or CHG sites, it is somewhat like "closing the door", which can easily inhibit the initiation of transcription. However, not every promoter methylation silences genes, and this cannot be generalized. On the contrary, non-CG methylations, such as CHG and CHH, are more like "sealing devices" in the genome, specifically targeting transposons and repeating elements to prevent them from moving around (Niederhuth and Schmitz, 2017). Once there is a problem with such methylation, such as dropping or being removed, those regions that should be silent may suddenly "speak out", thereby disrupting the normal expression rhythm of genes (Domb et al., 2020). What's more interesting is that methylation is not static; it fluctuates with the environment. For instance, under salt stress, the methylation levels of some stress-responsive genes will decline, thus being "unbound" and their expression levels will increase. This can help plants respond to external stress in a timely manner (Bartels et al., 2018). 4 Dynamic Methylation Patterns in Maize Roots Under Salt Stress 4.1 Salt stress treatment conditions and time-point design When studying the methylation response of corn roots, a common practice is to choose hydroponic or liquid culture systems as the experimental basis. Set the NaCl concentration between 100 and 200 mM. This range can simulate moderate to severe stress without causing the plant to die quickly. In some experiments, seedlings were treated with a 150 mM NaCl concentration and samples were taken every few hours (such as 1, 3, 6, 12, and 24 hours) in order not to miss the fluctuations in the early stage of methylation reaction (Fedorin et al., 2022). However, some people chose comparative treatment, such as two concentrations of 100 and 200 mM, and observed the differences in high and low salt environments. How to set the specific time point is often related to the goal: if you want to see a quick response, take samples intensively; if you want to see a long-term trend, extend the timeline. 4.2 Dynamic changes in methylation levels across different sequence contexts Not all methylations are affected by salt stress, but some patterns are quite obvious. For instance, in the three sequence environments of CG, CHG, and CHH, the positions of CHG and CHH are more likely to change under salt stress (Sun et al., 2018). Interestingly, after a short period of salt treatment, the overall m5C content decreased rapidly (He et al., 2024), suggesting that demethylation might be an initial reaction. In the promoter regions of some key genes (such as ZmEXPB2 and ZmXET1), especially in salt-sensitive corn, methylation levels are rapidly downregulated, while for those genotypes that are inherently more tolerant to salt, the methylation status appears more stable. Overall, DNA methylation is not a one-size-fits-all approach. Instead, it follows its own reaction logic depending on the genotype and region. 4.3 Identification of differentially methylated regions (DMRs) and association with target genes To identify which regions' methylation changes are most worthy of attention, high-throughput sequencing remains the most commonly used approach at present. In some studies, more than 4,400 differentially methylated regions (DMR) could be detected in salt-treated samples, and hypomethylation was more common than hypermethylation, especially under high-salt conditions (Sun et al., 2018). Most of these DMR are distributed in promoters, introns and even the flanking regions of genes, especially in genes related to cell metabolism and signal transduction (Tan, 2010). For instance, a negative regulatory gene like zmPP2C, which is involved in stress regulation, will have its expression suppressed once its intron is highly methylated. However, for genes like zmGST, their expression actually increases after demethylation. Both positive and negative examples demonstrate that there is indeed a clear correspondence between changes in methylation status and gene expression. 5 Identification of Key Genes and Pathways Regulated by DNA Methylation 5.1 Methylation features of transcription factors and functional genes responsive to salt stress Not all changes in gene expression under salt stress can be attributed to the regulation of transcription factors.
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