MsWRKY33 increases alfalfa (Medicago sativa L.) salt stress tolerance through altering the ROS scavenger via activating MsERF5 transcription.

Alfalfa (Medicago sativa L.) is considered to be the most important forage crop on a global scale. Nevertheless, soil salinity significantly decreases productivity, seriously threatening food security worldwide. One viable strategy is to explore salt stress-responsive factors and elucidate their underlying molecular mechanism, and utilize them in further alfalfa breeding. In the present study, we designated MsWRKY33 as a representative salt stress-responsive factor preferentially expressed in alfalfa roots and leaves. Subsequently, it was demonstrated that MsWRKY33 was localized in the cell nucleus, and functioned as a transcriptional activator of the W-box element. Transgenic alfalfa overexpressing MsWRKY33 displayed enhanced salt stress tolerance and antioxidant activities with no significant difference in other agronomic traits. Transcriptome profiling of MsWRKY33 transgenic alfalfa under control and salt treatment unveiled significantly altered expression of reactive oxygen species (ROS) scavenger genes in transgenic alfalfa. Subsequent examination revealed that MsWRKY33 binded to the promoter of MsERF5, activating its expression and consequently fine-tuning the ROS-scavenging enzyme activity. Furthermore, MsWRKY33 interacted with the functional fragment of MsCaMBP25, which participates in Ca signaling transduction. Collectively, this research offers new insight into the molecular mechanism of alfalfa salt stress tolerance and highlights the potential utility of MsWRKY33 in alfalfa breeding.