Background Late embryogenesis abundant (LEA) proteins are pivotal mediators of plant abiotic stress adaptation, but their functional roles and regulatory mechanisms in forage crops like Medicago sativa remain unclear. Through a genome-wide analysis of the LEA_3 subgroup in alfalfa, we identified MsLEA_3–6, whose expression is significantly induced by drought. To elucidate its role in drought tolerance, we conducted a comprehensive molecular and physiological characterization of MsLEA_3–6. Results Phylogenetic analysis placed MsLEA_3–6 within a conserved clade of drought-responsive LEA proteins. Its promoter harbored stress-related cis-elements, and its expression was upregulated under drought and salinity. Subcellular localization revealed plasma membrane association, while β-glucuronidase GUS staining confirmed ubiquitous expression in the roots, stems, leaves, flowers, and seeds of transgenic Arabidopsis. Overexpression of MsLEA_3–6 enhanced drought tolerance in Arabidopsis (improved root growth and germination under mannitol) and alfalfa (reduced H2O2 and malondialdehyde accumulation, elevated proline/soluble sugars, and attenuated oxidative stress via ROS scavenging). A soil moisture monitoring system demonstrated that MsLEA_3–6 overexpression led to improved photosynthetic performance under drought conditions. Furthermore, the expression of stress/ABA-responsive genes (ABI5, ABF3, NCED5, and NCED9) was altered in the transgenic lines compared with those in the wild-type plants under both normal and drought conditions. Dual-luciferase assays identified MsWRKY71 (MS.gene023126) as a direct binder of the MsLEA_3–6 promoter, forming a stress-responsive regulatory module. Conclusions Our findings identify MsLEA_3–6 as a functionally conserved, drought-responsive LEA protein that enhances osmotic adjustment and oxidative stress mitigation in transgenic plants, likely through coordinated regulation of ABA signaling and MsWRKY71-mediated transcriptional activation. While the findings support the functional relevance of MsLEA_3–6 under controlled conditions, further studies are needed to elucidate the underlying molecular mechanisms and to validate its utility in improving drought tolerance under field environments.

MsLEA_3–6 overexpression enhances drought tolerance in Medicago sativa L.

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2025年10月30日

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Wenxue Ma,
Xin Wen,
Lu Zhao,
Shuwei Dong,
Dong Luo,
Qiang Zhou,
Longfa Fang,
Wenxian Liu,
Zhipeng Liu

摘要

Background

Late embryogenesis abundant (LEA) proteins are pivotal mediators of plant abiotic stress adaptation, but their functional roles and regulatory mechanisms in forage crops like Medicago sativa remain unclear. Through a genome-wide analysis of the LEA_3 subgroup in alfalfa, we identified MsLEA_3–6, whose expression is significantly induced by drought. To elucidate its role in drought tolerance, we conducted a comprehensive molecular and physiological characterization of MsLEA_3–6.

Results

Phylogenetic analysis placed MsLEA_3–6 within a conserved clade of drought-responsive LEA proteins. Its promoter harbored stress-related cis-elements, and its expression was upregulated under drought and salinity. Subcellular localization revealed plasma membrane association, while β-glucuronidase GUS staining confirmed ubiquitous expression in the roots, stems, leaves, flowers, and seeds of transgenic Arabidopsis. Overexpression of MsLEA_3–6 enhanced drought tolerance in Arabidopsis (improved root growth and germination under mannitol) and alfalfa (reduced H₂O₂ and malondialdehyde accumulation, elevated proline/soluble sugars, and attenuated oxidative stress via ROS scavenging). A soil moisture monitoring system demonstrated that MsLEA_3–6 overexpression led to improved photosynthetic performance under drought conditions. Furthermore, the expression of stress/ABA-responsive genes (ABI5, ABF3, NCED5, and NCED9) was altered in the transgenic lines compared with those in the wild-type plants under both normal and drought conditions. Dual-luciferase assays identified MsWRKY71 (MS.gene023126) as a direct binder of the MsLEA_3–6 promoter, forming a stress-responsive regulatory module.

Conclusions

Our findings identify MsLEA_3–6 as a functionally conserved, drought-responsive LEA protein that enhances osmotic adjustment and oxidative stress mitigation in transgenic plants, likely through coordinated regulation of ABA signaling and MsWRKY71-mediated transcriptional activation. While the findings support the functional relevance of MsLEA_3–6 under controlled conditions, further studies are needed to elucidate the underlying molecular mechanisms and to validate its utility in improving drought tolerance under field environments.