Molecular Plant Breeding 2025, Vol.16, No.2, 105-118 http://genbreedpublisher.com/index.php/mpb 112 Additionally, highlighted the role of various TFs, including members of the NAC family, which are upregulated during water-deficit stress and are key regulators of multiple abiotic stresses. The study further identified 5 284 DEGs under drought stress, with many being tissue- or stage-specific, indicating a complex regulatory network that responds to water deficit conditions. DRG9 is a newly discovered drought-resistant gene that encodes a double-stranded RNA-binding protein. Under drought stress, DRG9 aggregates into stress granules through liquid-liquid phase separation, positively regulating the drought resistance of rice. The DRG9-DR allele is enriched in upland rice, and its introduction into cultivated varieties can significantly improve drought resistance (Wang al., 2024). Additionally, genes such as OsDREB1G, OsNAC6, OsLEA3, OsPP2C, DROT1, and qRT9 play key roles in rice's response to drought (Xu et al., 2016; Sun et al., 2022; Wang et al., 2024). Favreau et al. (2023) demonstrated gene expression and gene ontology (GO) enrichment analysis in rice under different genotypes and water treatment conditions. Under CIR-IRR and IAC-IRR conditions, genes for multiple biological processes and molecular functions, such as lignin metabolism, cell death, defense response, photosynthesis, etc., were significantly overexpressed, which were important biological processes in response to drought stress. The color of each network node represents the corrected p-value, yellow to orange indicates significant enrichment, and white nodes represent the parent node of the significant enrichment category. The histogram shows the mean gene expression for each sample and the significance levels for different genotypes and water treatments. Under drought conditions, rice adapts to a water-deficient environment by regulating several key genes. These genes are involved in defense response, hormone signaling, photosynthesis, and modification of cell structures, providing important information for understanding the mechanisms of drought response. 6.2 CRISPR-Cas9 mediated validation of candidate genes CRISPR-Cas9 technology has been employed to validate candidate genes involved in drought tolerance. The study identified several candidate genes such as OsOFP2, Dlf1, and OsMADS56, which were associated with limited water conditions. These genes were further validated using CRISPR-Cas9 to confirm their roles in drought tolerance. Similarly, Sakran et al. (2022) utilized genomic and transcriptomic approaches to identify and validate 36 DEGs with specific inherited alleles in the drought-resistant rice maintainer HuHan2B. These DEGs were involved in the regulatory network of TFs and target genes, demonstrating the potential of CRISPR-Cas9 in validating and improving drought tolerance traits. Another study focused on candidate genes such as OsDREB2A, OsNAC6, and OsPP2C. Using CRISPR-Cas9 technology, specific sgRNAs were designed to target specific sequences of these genes to validate their drought resistance functions. The knockout of the OsDREB2A gene led to significant changes in the gene expression profile of rice under drought conditions, especially the significant downregulation of gene expression related to drought response, indicating that OsDREB2Aplays a central role in transcriptional regulation under drought stress (Cui et al., 2011). The knockout of the OsNAC6 gene affected the root development and water absorption capacity of rice, weakening the plant's viability under drought conditions and further confirming the positive role of OsNAC6 in drought resistance (Nakashima et al., 2007). In summary, CRISPR-Cas9 technology provides an efficient and precise method for validating candidate genes for drought resistance. 6.3 Role of ABA and other hormones in gene regulation ABA and other hormones play crucial roles in the regulation of gene expression under drought conditions. The study identified TFs involved in both ABA-dependent and ABA-independent pathways that are upregulated during water-deficit stress (Ray et al., 2021). This includes members of the zinc-finger TFs and the NAC TF family, which are key regulators of abiotic stress responses. Additionally, Seeve et al. (2009) highlighted the involvement of ABA and auxin-dependent signaling pathways in regulating root growth responses to water deficits. Park et al.(2009) revealed the interaction between ABA receptors PYR/PYL/RCAR and PP2C protein phosphatases and SnRK2 protein kinases, which together regulate the physiological processes of stomatal closure and root growth, thereby enhancing the drought resistance of rice. Another study emphasized the differential expression of genes involved in hormone-mediated signaling and lignin biosynthesis, which is crucial for rice's tolerance response to osmotic stress (Baldoni et al., 2019). In addition to ABA and IAAs, GAs, ethylene (ETH), and cytokinins (CTK) also play important roles in plant responses to drought stress. For example, under drought
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