MsDREB2A modulates alfalfa growth by regulating MsCRE1 involved in cytokinin biosynthesis

Space mutagenesis is crucial for developing new plant breeds. We previously generated a rapidly growing mutant by inducing mutagenesis in 'Deqin' alfalfa seeds in outer space. However, the underlying molecular mechanisms remain unclear. Herein, we aimed to elucidate the molecular regulatory mechanisms that drive rapid alfalfa growth. We analyzed the ethylene response factor (ERF) gene family and identified the Medicago sativa cytokinin response element 1 (MsCRE1) gene, a critical regulator within the cytokinin signaling pathway. Functional validation revealed that overexpressing MsCRE1 in alfalfa and tobacco (Nicotiana tabacum) significantly enhanced plant growth rates and substantially increased cytokinin contents. We identified a close association between MsCRE1 and DREB2A of the AP2/ERF family in the alfalfa functional genomics database. Subcellular localization revealed that Medicago sativa Dehydration-responsive element binding protein 2A (MsDREB2A) is a nuclear-localized negative regulator, and we found that it specifically bound to MsCRE1. Dual-luciferase reporter assays revealed that MsDREB2A significantly activated MsCRE1 expression. Mechanistic studies indicated that MsDREB2A directly binds to the GGCGGTGGCGGTGG motif in the MsCRE1 promoter region. Downregulation of MsDREB2A via RNA interference significantly increased growth rates in both alfalfa and tobacco, whereas overexpression of MsDREB2A reduced growth in both species. We identified a molecular mechanism through which the MsDREB2A-MsCRE1 module controls vegetative growth in alfalfa via cytokinin signaling. This offers a promising target for molecular breeding programs.