Legume Genomics and Genetics 2026, Vol.17, No.1, 32-48 http://cropscipublisher.com/index.php/lgg 41 5.3 Methods for functional validation of candidate drought resistance genes Functional validation is indispensable for confirming the roles of candidate drought-responsive genes identified by transcriptomics, GWAS, or network analysis. In soybean, Agrobacterium-mediated transformation of composite plants with transgenic hairy roots is widely used for rapid gain- and loss-of-function assays. Overexpression of TF genes such as GmNAC3, GmNAC19, and GmHdz4 in hairy roots improved root system architecture, increased fresh and dry root biomass, and enhanced PEG-simulated drought tolerance, often accompanied by reduced hydrogen peroxide and superoxide accumulation and elevated antioxidant enzyme activities (Cui et al., 2024). Conversely, CRISPR/Cas9-mediated editing of GmHdz4 and other regulators demonstrated that disrupting negative regulators or fine-tuning TF dosage can significantly alter osmolyte accumulation, ROS detoxification, and survival under water deficit (Zhong et al., 2022; Khatamov et al., 2025). These root-focused systems are complemented by stable whole-plant transformants, in which phenotypes such as germination rate, plant height, leaf water status, and yield can be assessed across developmental stages and in field conditions. For example, overexpression of GmAP2/ERF144, GmERF205, and the LEA gene GmPM35 in soybean conferred higher relative water content, improved photosynthetic parameters, reduced MDA and ROS accumulation, and increased yield or biomass under drought (Wang et al., 2025). A wide range of physiological, biochemical, and molecular assays is used to quantify drought tolerance in validated lines. Standard measurements include relative water content, electrolyte leakage, chlorophyll content, proline and soluble sugar levels, MDA content, and activities of SOD, CAT, and POD, alongside NBT/DAB staining and gas-exchange parameters to monitor ROS status and photosynthetic efficiency (Wu et al., 2025). RT-qPCR is routinely employed to confirm transgene expression and to assess downstream targets predicted from regulatory networks, as in the case of GmAP2/ERF144 and GmNAC3 (Wang et al., 2022). Multi-omics validation is emerging, with some studies combining transcriptome-proteome integration or sRNA-degradome-RNA-seq to link gene perturbation with global network changes (Shahriari et al., 2022). New pipelines that integrate feature-engineering with co-functional networks (e.g., SoyNet) further prioritize candidates and pathways for experimental follow-up (Kao et al., 2025). Together, these methodological advances-ranging from rapid hairy-root assays and genome editing to field-level phenotyping and multi-omics integration-are progressively converting computational predictions into mechanistically grounded targets for breeding drought-resilient soybean. 6 Case Study: Transcriptomic Analysis of Key Genes Involved in Soybean Drought Stress Response 6.1 Transcriptome comparison of soybean varieties with varying drought tolerance Comparative transcriptomic studies using contrasting soybean genotypes have clarified how tolerant and sensitive varieties deploy distinct molecular strategies under drought. At the seedling stage, drought-tolerant Jindou 21 (JD) and drought-sensitive Tianlong No.1 (N1) displayed markedly different DEG profiles: 6038 DEGs were detected in JD versus 4112 in N1, indicating a broader and more dynamic transcriptional reprogramming in the tolerant cultivar (Xuan et al., 2022). KEGG enrichment showed that JD preferentially activated plant hormone (JA, brassinosteroid) signaling, calcium and MAPK cascades, and stress-related TFs and cell-wall remodeling genes, whereas these pathways were weaker or absent in N1 (Xuan et al., 2022). A similar pattern was found in wild soybean, where tolerant genotypes (DTP, DTL) and sensitive genotypes (DSP, DSL) showed 4850 and 6272 DEGs, respectively, but only 547 DEGs had consistent opposite expression between tolerant and sensitive groups, highlighting a core set of genes whose genotype-dependent regulation underpins drought tolerance at germination. At later stages, comparative analyses of elite cultivars under gradual water deficit or PEG-induced drought further revealed variety-specific strategies. The drought-tolerant Heinong 44 (HN44) maintained higher ABA levels, smaller stomata, lower stomatal conductance, and higher instantaneous water-use efficiency than the sensitive Suinong 14 (SN14), supported by transcriptomic enrichment of ABA signaling and glutathione metabolism in HN44 under severe stress (Xu et al., 2023). In another seedling study, drought-resistant L14 exhibited a “drought-avoidance” strategy with slow-wilting phenotype, fewer DEGs and relatively stable photosynthesis, carbohydrate and lipid metabolism compared with drought-sensitive L21, which showed stronger transcriptional
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