Bioscience Methods 2025, Vol.16, No.6, 280-288 http://bioscipublisher.com/index.php/bm 281 basis for future breeding strategies and biotechnology intervention measures to enhance the drought tolerance of rye and other crops, and to ensure global food security under climate change conditions. 2 Physiological and Molecular Responses of Rye to Drought Stress 2.1 Effects of drought stress on rye growth and development Rye often performs worse than expected in drought years, especially with significant limitations in grain yield, biomass and root development (Bao et al., 2022). Different test results show that the yield reduction can sometimes reach 57%, but there are also varieties that have only decreased by about 14%. In fact, not only the yield, but also multiple developmental stages such as the number of tillers, leaf spreading, and root extension will be disturbed (Kottmann et al., 2016; Makhramova and Urokov, 2024). Some varieties will show the phenomenon of early maturity, and the photosynthetic area will also decrease accordingly. Physiologically, water loss, decreased photosynthetic capacity, and hormonal imbalances (especially the reduction of trans zeaxin nucleosides) all make it difficult for plants to maintain normal growth (Vedenicheva et al., 2024). However, not all rye is the same. Some varieties with special genetic backgrounds can still maintain relatively stable productivity when water is scarce, indicating that there are certain genetic differences in drought resistance. 2.2 Drought-related signal transduction pathways in rye When rye encounters drought, it does not simply act passively but activates a complete set of signal transduction mechanisms within its body. ABA (abolic acid) is often the earliest mentioned regulatory hormone. Its accumulation can activate a series of genes that respond to drought, such as those encoding serine/threonine protein kinases (Tomita et al., 2021; Movahedi et al., 2023). But in fact, apart from ABA, cytokinin and ROS (reactive oxygen species) are also at play. They regulate growth and defense responses to a certain extent. Some transcription factors, such as WRKY, DREB and NAC, are significantly upregulated under drought conditions, and this expression change can link multiple stress response genes (Cheng et al., 2022). This entire pathway will eventually control a series of processes such as stomatal closure, osmotic regulation, and antioxidant reactions, helping rye alleviate the impact of drought. 2.3 Overview of drought resistance traits and gene expression regulation networks The drought tolerance of rye is rarely determined by a single gene. It is usually the result of the combined effects of root structure, antioxidant levels and metabolic homeostasis. At the molecular level, researchers have identified thousands of differentially expressed genes and proteins under different conditions, involving pathways such as energy metabolism, lipid synthesis, and signal transduction (Pan et al., 2017; 2018). What regulates these changes is a vast regulatory network. Among them, there are transcription factors and protein kinases, as well as E3 ligases involved in the ubiquitination process. Together, they regulate ABA signaling and its related stress pathways (Wang et al., 2023). These genes and networks are associated with some obvious drought-resistant traits, which can provide a clear direction for the breeding of drought-tolerant rye varieties. 3 Basic Features and Functional Mechanisms of microRNAs 3.1 Biosynthesis and modes of action of microRNAs The synthesis process of miRNA seems like a complex assembly line, from gene transcription to the loading of mature miRNA into protein complexes, each step is not simple. miRNA usually consists of only 20 to 24 nucleotides and is an endogenous non-coding RNA that plays the role of a regulator in plants. They were initially synthesized into pri-miRNA by RNA polymerase II, and this precursor folds into a stem-ring structure (Zhang and Wang, 2025). DCL1 is the core enzyme involved in the processing. Meanwhile, HYL1 and SERRATE also get involved, gradually processing pri-miRNA into pre-miRNA and then transforming it into mature miRNA/miRNA double-stranded entities. This process all takes place in the cell nucleus. When the mature miRNA is ready, it will be methylated, then loaded into the AGO protein to form the RISC complex, and then transported to the cytoplasm to continue "functioning" (Figure 1) (Wang et al., 2019). 3.2 Post-transcriptional regulatory mechanisms mediated by microRNAs Once miRNA enters the RISC complex, it does not directly control the transcription of genes. Instead, it "targets" mrnas that are highly complementary to its own sequence, usually making a cut at these targets. After the cut, the
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