Legume Genomics and Genetics 2025, Vol.16, No.5, 225-233 http://cropscipublisher.com/index.php/lgg 231 6.3 Functional studies using epigenetic mutants Some researchers have directly conducted experiments on whether these regulatory factors are "necessities". For instance, by knocking down DME through RNAi, problems in the differentiation process of root nodules can be observed-on the one hand, DNA methylation increases; on the other hand, the expression of key genes is suppressed (Satge et al., 2016). Not only that, the use of CRISPR-Cas9 to disrupt the function of DNA methyltransferase MtDRM2 can also cause impaired methylation of CHH and abnormal root tumor development, which further indicates that the balance between methylation and demethylation is indispensable. However, if the enzymes knocked down are plant-specific enzymes related to histone modification such as HDAC or HAT, it will also be found that the meristematic tissue shrinks and bacterial colonization decreases. Based on these results, both methylation at the DNA level and modification at the histone level have a direct impact on the formation and functional performance of root nodules. 7 Future Directions 7.1 Epigenome editing to enhance legume-rhizobia symbiosis Most of the existing gene editing technologies focus on the alteration of DNA sequences. However, in some cases, the genes themselves remain unchanged, but their expression can be regulated-this is precisely where epigenome editing comes into play (Baldini et al., 2025). For instance, if the CRISPR/dCas9 system is combined with chromatin modification enzymes, it can precisely "mute" or "activate" certain genes. Transcription factors like NIN that control nodulation or chromatin remodeling factors that intervene in the initiation process of symbiosis are potential targets. However, such tools are not "universal keys". The genetic backgrounds of different leguminous plants vary greatly. To truly achieve wide application, it is necessary to first solve the problems of target selection and adaptation of transformation platforms. Future research is very likely to start from these two aspects. 7.2 Breeding for epigenetic traits in legume crops Traditional breeding methods do not always meet the current agricultural demands in terms of efficiency, especially against the backdrop of increasingly severe climate change. Epigenetic variations, although once overlooked, are actually playing an increasingly significant role in plants' adaptation to stress and trait differentiation. Take leguminous crops as an example. Those methylation patterns or epigenetic "switch genes" that can be inherited may directly affect the number, size and even nitrogen fixation efficiency of root nodules. In the past, we often used QTL as markers. Now, epiQTL (epigenetic quantitative trait loci) can also be considered to assist in selection. Of course, relying solely on this approach is not enough. Only by integrating epigenetic selection with conventional breeding and genomic selection is it more likely to truly accelerate the pace of variety improvement. 7.3 Multi-Omics integration in nodule development and epigenetics To understand how root nodules develop step by step, relying solely on a single omics is often insufficient. After all, epigenetic regulation itself involves multiple levels, such as DNA methylation, transcriptional regulation, protein translation, and metabolic status, all of which may influence each other. Integrating epigenomics, transcriptomics, proteomics and even metabolomics data, although complex, has become a trend. More advanced methods, such as single-cell multi-omics, can help us break down the state differences among various cell types. Time series analysis, on the other hand, can track the entire process of regulatory events from their occurrence to their fading (Pereira et al., 2024). Ultimately, we hope that with these data, we can not only explain a certain phenomenon, but also reconstruct the entire regulatory network regulated by epigenetic mechanisms, and even predict the response patterns of root tumor development in different environments. 8 Concluding Remarks In the relationship between plants and microorganisms, some regulatory mechanisms are not so prominent, but their significance cannot be ignored. Epigenetic regulation is one of them. Especially in the symbiotic system of leguminous plants and rhizobia, it is no longer a "minor role", but has gradually become a key level in the entire
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