Abstract:

【Background】Drought stress is one of the major abiotic factors limiting global agricultural productivity. Systematic elucidation of transcriptional regulatory mechanisms during drought and rehydration processes is crucial for improving crop drought tolerance and advancing molecular breeding strategies. Medicago ruthenica Sojak cv. Zhilixing, a perennial leguminous forage species, exhibits strong ecological adaptability and drought resistance.【Objective】This study aimed to identify key regulatory modules and core functional genes involved in drought and rehydration responses through transcriptome analysis and co-expression network construction, thereby revealing the underlying molecular mechanisms. 【Method】Four treatment stages were established, including normal irrigation (Group A), mid-term drought stress (Group B), late-stage drought stress (Group C), and post-rehydration (Group D), to simulate the transcriptional response of M. ruthenica under progressive drought and recovery conditions. High-throughput RNA sequencing was performed to obtain gene expression profiles, followed by weighted gene co-expression network analysis (WGCNA) to construct expression modules. Principal component analysis (PCA) and KEGG pathway enrichment were conducted to assess expression variation and functional clustering. Modules and hub genes closely associated with drought tolerance were identified, and six representative genes from the MEmagenta and MEdarkgreen modules were selected for qRT-PCR validation to assess data consistency and biological relevance. 【Result】PCA revealed clear separation of samples across PC1 and PC2 dimensions, indicating stage-specific effects of drought and rehydration treatments on gene expression patterns. Differential expression analysis showed the greatest number of up- and down-regulated genes during mid-stage drought (Group B), suggesting rapid activation of stress- responsive mechanisms. In the late-stage drought (Group C), fewer differentially expressed genes were observed, while pathways related to fatty acid degradation and carbohydrate metabolism were significantly enriched, suggesting a shift toward homeostatic regulation. During rehydration (Group D), most gene expression levels gradually recovered, although some signaling and defense pathways remained active, indicating ongoing adaptive modulation.WGCNA identified four modules significantly correlated with specific treatments (|r| > 0.6). The MEdarkgreen module (r = 0.93), highly expressed in Group C, was enriched in MAPK signaling, endoplasmic reticulum (ER) stress response, lipid metabolism, and flavonoid biosynthesis. Hub genes BZIP17 and IRE1B were implicated in ER stress signaling, protein folding regulation, and transcriptional reprogramming, indicating their key roles in prolonged drought adaptation. The MEmagenta module (r = 0.82), highly expressed under normal conditions (Group A), was enriched in ABA and JA signaling pathways, as well as flavonoid metabolism. Its core genes, ABF4 and MYC2, are involved in stomatal regulation and secondary metabolic control, functioning as crucial regulators during the early drought response. Additional genes such as NAC072 and CLPD contribute to chloroplast protein stability and reactive oxygen species scavenging, supporting their multifaceted roles in stress mitigation. KEGG enrichment results were highly consistent with module functions, confirming the reliability of functional annotations. qRT-PCR analysis showed that all six selected genes were significantly upregulated during mid and late drought stages (Groups B and C) and downregulated upon rehydration (Group D), mirroring RNA-seq expression patterns and validating the biological relevance of the identified modules and genes. 【Conclusion】This study reveals dynamic transcriptional regulation patterns in M. ruthenica under drought stress and rehydration. Two key modules, MEmagenta and MEdarkgreen, were identified as strongly associated with drought adaptation, encompassing hub genes such as ABF4, MYC2, BZIP17, and IRE1B. These genes play central roles in signal transduction, metabolic adjustment, and stress response, representing core components of the molecular mechanisms underlying drought adaptation in M. ruthenica. The findings provide a theoretical foundation and candidate targets for elucidating drought-responsive pathways and advancing molecular breeding of drought-resilient forage crops.

Key words: Medicago ruthenica, transcriptome, weighted gene co-expression network analysis (WGCNA), drought resistance, key genes