MsDAD1 acts as a heat-induced “senescence brake” in alfalfa

Heat stress severely limits the productivity of alfalfa (<i>Medicago sativa</i> L.). In this study, the defender against apoptotic death 1 (<i>DAD1</i>) gene, <i>MsDAD1</i>, was identified and functionally characterized as a key positive regulator of heat tolerance. The expression of <i>MsDAD1</i> was specifically and strongly induced by heat stress, and phylogenetic analysis confirmed its high conservation across plant species. Ectopic overexpression of <i>MsDAD1</i> in transgenic alfalfa significantly enhanced tolerance to heat stress. Compared to wild-type plants, <i>MsDAD1</i>-overexpressing lines (<i>MsDAD1</i>-OE) exhibited reduced leaf chlorosis and abscission, higher relative water content, lower electrolyte leakage, greater chlorophyll retention, and diminished accumulation of reactive oxygen species (H₂O₂, O₂ ^-) and malondialdehyde (MDA), suggesting improved membrane integrity and reduced oxidative damage. Transcriptome (RNA-seq) analysis and subsequent physiological validation indicated that <i>MsDAD1</i> suppresses heat-induced accumulation of jasmonic acid (JA) and abscisic acid (ABA) by down-regulating key biosynthetic genes, <i>LOX1</i> and <i>NCED1</i>. As a result, <i>MsDAD1</i>-OE plants displayed attenuated JA- and ABA-mediated leaf senescence under heat stress. Furthermore, <i>MsDAD1</i> overexpression delayed heat-induced flowering, correlating with the repression of flowering-promoting genes such as <i>FT</i> and <i>ELF4</i>. Collectively, these findings demonstrate that <i>MsDAD1</i> enhances alfalfa heat tolerance by mitigating oxidative stress, modulating JA and ABA biosynthesis to delay senescence, and altering flowering time under high-temperature conditions. <i>MsDAD1</i> represents a promising genetic target for improving heat resilience in alfalfa.