Bioscience Methods 2025, Vol.16, No.6, 280-288 http://bioscipublisher.com/index.php/bm 286 resistance. For instance, it can influence abscisic acid (ABA) signaling, eliminate reactive oxygen species (ROS), and shape root structure, among other important processes. Drought-responsive mirnas such as miR156, miR159, miR164, miR172 and miR398 usually target transcription factors and some stress-related genes. A complete set of complex regulatory networks (miR156, miR159, miR164, miR172 and miR398) has been constructed. The identification of these mechanisms is attributed to high-throughput sequencing and integrated bioinformatics tools, which enable us to more systematically identify the existence and targets of these mirnas, thereby advancing our understanding of post-transcriptional regulation in cereal crops and laying the foundation for breeding applications. However, the research has not been smooth sailing all the way, especially when it comes to truly applying these achievements to breeding, there are still many obstacles to overcome. For instance, under different genotypes and experimental conditions, the expression patterns of the same miRNA often vary, and the tissue specificity is also obvious, which makes the induction of "universal mirnas for drought response" quite challenging. Furthermore, although targets can now be predicted through tools like psRNATarget and TargetFinder, and verified by means such as RACE-PCR, qRT-PCR, and degradation omics sequencing, the entire process has become relatively mature. However, the operational costs and data processing pressure remain considerable. One more point that is often overlooked is that species like rye, which are valuable in agriculture but have received little research, also urgently need to be followed up. We should not always focus on a few model plants. New technologies such as single-cell sequencing and AI prediction may be breakthroughs to enhance the accuracy of miRNA annotation and functional recognition, but at present, they are still in their infancy. By the way, the functional research of miRNA has still opened up many new paths for crop improvement. Mirna-based methods such as overexpression, knockdown, and CRISPR/Cas9 editing have already achieved "good data" on some model plants and major food crops. Whether similar effects can be achieved on rye in the future depends on the specific implementation. Judging from the current trend, whoever can safely incorporate the experimental results into the breeding system will be able to add an extra layer of security to food security in the context of increasingly frequent droughts. Acknowledgments We would like to express our gratitude to the reviewers for their valuable feedback, which helped improve the manuscript. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Ajila V., Colley L., Ste-Croix D., Nissan N., Golshani A., Cober E., Mimee B., Samanfar B., and Green J., 2023, P-TarPmiR accurately predicts plant-specific miRNA targets, Scientific Reports, 13: 4005. https://doi.org/10.1038/s41598-022-27283-8 Aravind J., Rinku S., Pooja B., Shikha M., Kaliyugam S., Mallikarjuna M., Kumar A., Rao A., and Nepolean T., 2017, Identification characterization and functional validation of drought-responsive MicroRNAs in subtropical maize inbreds, Frontiers in Plant Science, 8: 868. https://doi.org/10.3389/fpls.2017.00941 Bao G., Pan X., Yan B., Chang Y., Tang W., Qu Y., Wei J., and Zhao H., 2022, Resistance of rye seedlings to drought and freeze-thaw stress, Polish Journal of Environmental Studies, 31(2): 1559-1568. https://doi.org/10.15244/pjoes/142608 Cheng S., Yang X., Zou L., Wu D., Lu J., Cheng Y., Wang Y., Zeng J., Kang H., Sha L., Fan X., Ma X., Zhang X., Zhou Y., and Zhang H., 2022, Comparative physiological and root transcriptome analysis of two annual ryegrass cultivars under drought stress, Journal of Plant Physiology, 277: 153807. https://doi.org/10.1016/j.jplph.2022.153807 Ding N., and Zhang B., 2023, microRNA production in Arabidopsis, Frontiers in Plant Science, 14: 1096772. https://doi.org/10.3389/fpls.2023.1096772 Dong Q., Hu B., and Zhang C., 2022, MicroRNAs and their roles in plant development, Frontiers in Plant Science, 13: 824240. https://doi.org/10.3389/fpls.2022.824240 Ferdous J., Hussain S., and Shi B., 2015, Role of microRNAs in plant drought tolerance, Plant Biotechnology Journal, 13: 293-305. https://doi.org/10.1111/pbi.12318
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