Maize Genomics and Genetics 2025, Vol.16, No.6, 325-333 http://cropscipublisher.com/index.php/mgg 329 Sometimes, the transformation of DNA methylation status seems more like the driving force behind the scenes. Take ZmKTF1 as an example. It plays a core role in the RNA-mediated DNA methylation (RdDM) pathway. Once a mutation occurs, the CHH methylation level will decline, and then it will affect the genes related to oxidoreductase activity throughout the body, thereby altering the accumulation of ROS and the salt tolerance of plants. In addition, the expressions of the "familiar faces" of transcription factor families such as WRKY, MYB, bZIP, and bHLH can also be affected by the methylation status (Liu et al., 2022; Zhang and Xu, 2024). For instance, research has found that under salt stress, the methylation of the first intron of the zmPP2C gene is enhanced, thereby suppressing the expression of this negative regulatory factor. However, genes like zmGST are upregulated in expression after demethylation, which has a positive effect on stress responses. These cases demonstrate that in a salt environment, transcriptional regulation also depends on the "face" of methylation, and the patterns vary greatly among different genes and in different environments. 5.2 Hormonal pathways and ROS signaling networks affected by methylation Under salt stress conditions, the signaling pathways of hormones and ROS are almost simultaneously activated, and epigenetic factors are often involved behind them. The expression differences of multiple key genes involved in hormone pathways such as ABA, auxin, and jasmonic acid are sometimes not dominated by transcription factors but caused by fluctuations in DNA methylation levels (Zhang et al., 2021; Ying et al., 2025). For example, ZmKTF1 is considered to be able to indirectly control ROS levels by regulating the expression of antioxidant genes, thereby affecting salt tolerance (Wang et al., 2024). The methylation changes of the promoters of some peroxidase and superoxide dismutase genes are also believed to be directly related to the efficiency of ROS clearance. Moreover, this regulation often does not occur alone - sequences modified by differential methylation can also frequently be found in ABA and auxin response genes, indicating that the linkage between hormones and ROS has already been "predefined" at the epigenetic level. This coupling mechanism might be a key for corn to maintain cellular homeostasis in a high-salt environment. 5.3 Functional enrichment analysis of upregulated/downregulated genes (GO and KEGG) Sometimes, if one wants to know exactly which genes were activated under a certain stress and what these genes did, directly looking at the enrichment results of GO and KEGG can often provide many clues. Among the differentially expressed genes, whether activated or inhibited, most are concentrated in functional blocks such as signaling pathways, secondary metabolism, MAPK, hormone responses, and phenylpropane metabolism. These modules do not exist in isolation; many of them work together under coercive circumstances. As for GO, terms such as "abiotic stimulus response", "REDOX enzyme activity", and "signal transduction" appear frequently, which also indicates from another perspective that DNA methylation regulation is indeed linked to stress adaptation (Maimaiti et al., 2025). However, not all pathways are so "directly related". Conventional metabolic pathways such as photosynthesis, carbohydrate metabolism, and amino acid synthesis, although they sound more like what is needed during the growth stage, were also listed by KEGG analysis (He et al., 2024). It might be the stable operation of these "infrastructures" that supports the overall regulatory capacity of plants in the face of salt stress. So, these regulatory pathways may seem complex, but in fact, under the regulation of DNA methylation, they have pieced together an interdependent system framework. 6 Case Study: Comparative Methylome Analysis of Salt-Tolerant and Salt-Sensitive Maize Varieties 6.1 Selection of varieties and verification of physiological response differences Before conducting methylation research, the selection of comparison materials actually determines half of the direction of the research. The AS5 and SPL02 strains are often regarded as representatives of salt tolerance because under multiple experimental conditions, they all demonstrate stronger salt resistance than NX420 or Mo17 - not only are there significant differences in biomass, but also obvious advantages in Na+ accumulation and water retention capacity (Zhu et al., 2023). However, not all salt-tolerant varieties perform stably well at different salt concentrations. Under the treatment of 150-180 mM NaCl, those materials that perform "outstandingly" can often still maintain relatively normal growth, while the leaves of sensitive materials show
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