MfWRKY40 Positively Regulates Drought Tolerance in Arabidopsis thaliana by Scavenging Reactive Oxygen Species

Drought stress is a major abiotic constraint that severely restricts the growth of <i>Medicago falcata</i> L. by inducing the accumulation of reactive oxygen species (ROS) in plants. WRKY transcription factors (TFs) play a key role in regulating plant responses to drought stress. In this study, we investigated the role of the <i>MfWRKY40</i> gene in drought tolerance. Under mannitol and ABA stress treatments, <i>MfWRKY40</i>-overexpressing lines (OEs) showed significantly longer primary roots, increased lateral roots, and higher fresh weight compared to wild-type (Col) lines, indicating significantly enhanced growth and drought tolerance. Similarly, under soil drought conditions, transgenic <i>Arabidopsis thaliana</i> exhibited enhanced drought tolerance. NBT staining demonstrated decreased ROS accumulation in transgenic lines after stress treatment. Correspondingly, the <i>MfWRKY40</i>-overexpressing lines displayed significantly lower levels of hydrogen peroxide (H₂O₂), superoxide anion (O₂^-), and malondialdehyde (MDA) compared to Col, along with elevated activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as increased proline (Pro) content. Furthermore, <i>MfWRKY40</i> upregulated the expression of antioxidant enzyme genes (<i>AtPOD3</i>, <i>AtSOD4</i>, and <i>AtCAT1</i>) and modulated the expression of other drought-related genes. In summary, our results demonstrate that <i>MfWRKY40</i> enhances drought tolerance in <i>A. thaliana</i> by improving ROS scavenging capacity. This study provides a theoretical foundation for further exploration of <i>MfWRKY40</i>'s functional mechanisms in drought stress adaptation.