Heterologous expression of MfWRKY40 enhanced drought resistance of alfalfa

Drought stress severely affects alfalfa growth. WRKY transcription factors play an important regulatory role in plant response to abiotic stresses. The aim of this study was to explore the role of MfWRKY40 in drought tolerance in alfalfa. We generated transgenic alfalfa plants overexpressing MfWRKY40 via Agrobacterium -mediated transformation and measured the expression levels in different lines by quantitative real-time PCR (qRT-PCR). Under natural drought stress, MfWRKY40 -overexpressing lines displayed improved phenotypic performance relative to wild type (WT) plants. The transgenic lines also exhibited reduced water loss rates and smaller stomatal apertures compared to WT plants. Analysis of photosynthetic parameters further revealed enhanced photosynthetic capacity in the transgenic plants. Additionally, under drought stress, antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities were significantly elevated in the transgenic alfalfa, while relative electrolytic leakage (REL), malondialdehyde (MDA) and superoxide anion (O 2 - ) content were significantly lower than that of WT plants. Furthermore, expression levels of enzymes and drought-responsive genes were higher in transgenic alfalfa than in the WT controls. Our findings indicate that MfWRKY40 improves drought tolerance in alfalfa by limiting water loss, enhancing photosynthetic performance, reducing reactive oxygen species (ROS) accumulation, and modulating the expression of enzymes and drought-responsive genes. The MfWRKY40 gene could be used as a molecular tool for breeding drought-tolerant alfalfa cultivars.