Bioscience Methods 2025, Vol.16, No.6, 280-288 http://bioscipublisher.com/index.php/bm 285 5.3 Expression patterns and signal integration of target genes in drought response Not all mirnas under drought stress are "completely suppressed". Some upregulated mirnas push down the target genes involved in growth and signaling pathways, which is a kind of "throttling" response. However, some mirnas themselves were inhibited by drought, and as a result, their target genes could be expressed more. This has become another drought resistance strategy (Shamloo-Dashtpagerdi et al., 2023). This is not just a matter between miRNA and target genes; there are also a bunch of signaling pathways such as ABA and ROS involved, making the entire regulatory system more refined. For example, miR408 in ryegrass, the target genes it controls enhance the drought tolerance of plants by affecting leaf structure and antioxidant function (Hang et al., 2020). Overall, the system composed of miRNA and target genes is like a complete command system for plants to mobilize their drought resistance response. At critical moments, it can also integrate different signal sources for overall coordination. 6 Case Studies: Roles of Key miRNAs in Rye Drought Tolerance 6.1 Functional analysis of miR398 in regulating the ROS scavenging system When it comes to how plants respond to oxidative stress under drought conditions, miR398 is basically an unavoidable role. It is quite "ancient" and conservative among many plants, but when it really works, it depends on how its expression state changes. Under drought conditions, miR398 is often not elevated but inhibited. As a result, the target genes it originally controlled, such as copper/zinc superoxide dismutase (CSD1 and CSD2), start to be upregulated. Such enzymes happen to be the main force in eliminating ROS and are quite crucial for maintaining the stability of the cellular environment (Li et al., 2022). Of course, this kind of regulation is not the more the better. Instead, it is a dynamic "delegation of power" behavior carried out by the plant itself within its regulatory system, with the aim of "speeding up" the antioxidant system. Many studies, including cases of crops such as grains, have demonstrated a point: regulating miR398 can indeed help plants enhance their "self-protection" ability to some extent when facing drought. 6.2 miR159-mediated regulation of the ABA signaling pathway Small Rnas like miR159 are also frequently mentioned in the mechanisms by which plants respond to drought. Its activity level will change with the occurrence of drought or the increase of ABA levels. Interestingly, although it is induced by ABA, it in turn acts on some core downstream links of ABA, especially regulating the transcription factors of the MYB family, such as MYB33, and MYB33 in turn affects the expression of key response genes like ABI5. It's a bit convoluted, but the logic is clear: drought → increased ABA → increased miR159 expression → suppressed MYB33 → ABI5 regulates adaptive response (Singroha et al., 2021; Jiang et al., 2022). In Arabidopsis thaliana, if the activity of miR159 is artificially reduced, the drought resistance of the plants becomes even stronger, indicating that this pathway does have a certain "braking" effect. This regulatory chain has also found similarities in cereal crops. As for whether it operates in the same way in rye, more experiments are needed for further confirmation at present. 6.3 Role of miR166 in leaf development and water retention As for miR166, its main focus is not on ROS or hormone pathways, but on leaf development. It affects the morphology and structure of leaves by regulating the expression of HD-ZIP Class III transcription factors, and thereby indirectly influences the plant's ability to retain water. Studies have found that after reducing the activity of miR166, the leaf morphology of plants changed and the transpiration rate slowed down, all of which were related to the improvement of drought tolerance (Singh et al., 2022). HD-ZIP III is not only related to vascular development but also to leaf polarity. Therefore, the regulatory range of miR166 is actually quite broad. At present, this miRNA has considerable potential in regulating the water use efficiency of plants under drought conditions, especially in crops like rye that have high requirements for environmental adaptation, which is even more worthy of in-depth research (Lang et al., 2025). 7 Conclusions and Future Perspectives When it comes to how plants survive drought, the role of miRNA cannot be bypassed. There have been numerous studies in the past, and it is widely recognized that miRNA plays a crucial role in regulating plant drought
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