The alfalfa (Medicago sativa L.) genotypes used in this study included the wild-type cultivar SY4D and MsDAD1 transgenic lines (OE#1 and OE#3), which were generated in the SY4D background. Rooted stem cuttings of both wild-type and transgenic plants were prepared and transplanted into 10×10 cm pots. Plants were grown under controlled environmental conditions: a 16/8 h light/dark photoperiod, a temperature of 23°C, relative humidity of 50–70%, and a light intensity of 300 μmol m^-² s^-¹.

To analyze the expression pattern of MsDAD1 under heat stress, four-week-old alfalfa seedlings were transferred to a climate chamber and exposed to high-temperature treatment (40°C) for 0, 1, 3, 6, 12, 24, and 48 hours. To evaluate the heat tolerance function of MsDAD1, transgenic and wild-type plants were subjected to a controlled heat stress regime in a growth chamber set at 32°C (night)/40°C (day), allowing for plant survival and the assessment of physiological responses. Phenotypic evaluations were conducted after six days of treatment.

For flowering time analysis under both normal and heat stress conditions, above ground part of wild-type and MsDAD1-overexpressing (OE) plants were cut off at the same time. Plants were divided into two groups: one maintained under normal conditions (20°C night/23°C day), and the other exposed to a heat stress regime of 30°C (night)/35°C (day), with identical photoperiod and light intensity settings as the control group.

For hormone treatment assays, healthy mature leaves were divided into two groups. One group was placed on half-strength Murashige and Skoog (½ MS) medium (control), and the other was treated with 50 μM jasmonic acid (JA) or abscisic acid (ABA), respectively. Unless otherwise stated, all experiments were performed with at least three biological replicates.

The full-length coding sequence (CDS) of MsDAD1 was amplified using gene-specific primers listed in Supplementary Table S1 . PCR amplification was performed with an initial denaturation at 98°C for 3 minutes, followed by 35 cycles of 98°C for 10 seconds, 55°C for 30seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. The amplified product was ligated into the pMD19-T vector (Takara) and verified by Sanger sequencing. Homologous polypeptide sequences of DAD1 were retrieved from the GenBank database. Phylogenetic analysis was conducted using the neighbor-joining method. Sequence alignment was performed with ClustalX (www.clustal.org), and the phylogenetic tree was constructed using MEGA version 6.0 (www.megasoftware.net) with 1000 bootstrap replicates to assess branch support and reliability.

Total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China), following the manufacturer’s protocol. First-strand cDNA synthesis was performed using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China). Quantitative real-time PCR (qRT-PCR) was conducted using 2× SYBR Green Mix (Vazyme, Cat. No. Q711-03) according to the manufacturer’s instructions. The ACT2 gene was used as an internal reference for normalization. Relative transcript levels were calculated using the 2^{^–ΔΔCt} method (Livak and Schmittgen, 2001). Primer sequences are listed in Supplementary Table S1 . Each biological sample was analyzed in triplicate.

To generate transgenic alfalfa seedlings, the coding sequence of MsDAD1 was cloned and inserted into the binary vector 35S::NOS::1300, under the control of the CaMV:: 35S promoter. The resulting construct was introduced into alfalfa via Agrobacterium tumefaciens-mediated transformation, following the protocol described in Transgenic Plants: Methods and Protocols (Jiang et al., 2019). Transgenic lines were selected on hygromycin-containing medium, and successful integration of MsDAD1 was confirmed by PCR analysis.

Relative water content (RWC) was determined using the leaf saturation method. Electrolyte leakage (EL) was measured with a conductivity meter (BELL, BEC-6600, Dalian, China). Briefly, six fully expanded, healthy leaves were collected from the middle canopy of each plant at the same developmental stage. The fresh weight of each sample was recorded prior to analysis and then incubated in 25 mL of double-distilled water. After 2 hours of gentle shaking, the initial conductivity of the solution was measured using a DIST-5 conductometer (Hanna Instruments). Samples were then boiled to release all electrolytes, and the final conductivity was recorded. EL was expressed as a percentage of the total conductivity and normalized to fresh weight. Chlorophyll content was measured using a SPAD chlorophyll meter (SPAD-502; Konica Minolta Sensing, Japan). Hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and superoxide anion (O₂ ^-) levels were quantified using commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China). Endogenous levels of jasmonic acid (JA) and abscisic acid (ABA) were quantified using 20 mg of fresh plant tissue. Phytohormones were extracted with 10% (v/v) methanol in water (MeOH/H₂O). A cocktail of stable isotope-labeled internal standards was added to validate the liquid chromatography–mass spectrometry (LC-MS) quantification. The extracts were purified using Oasis hydrophilic-lipophilic balanced (HLB) columns (30 mg/1 mL; Waters), and targeted analytes were eluted with 80% (v/v) methanol. The eluent, containing both neutral and acidic compounds, was gently evaporated to dryness under a stream of nitrogen. Chromatographic separation was carried out using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters) equipped with an Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm; Waters). The effluent was introduced into the electrospray ionization (ESI) source of a Xevo TQ-S triple quadrupole mass spectrometer (Waters) for targeted quantification of JA and ABA.

Two-week-old wild-type and MsDAD1-overexpressing (OE) seedlings were cultivated as previously described. Leaf tissues were harvested and immediately frozen in liquid nitrogen for total RNA extraction. For each sample, 1.5 μg of mRNA was used as input for library preparation. RNA sequencing libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. RNA concentration was measured using a NanoDrop 2000C spectrophotometer (Thermo Scientific, Mississauga, Canada), and RNA integrity was assessed with an Agilent 2100 Bioanalyzer using an RNA Nano chip (Agilent Technologies, Santa Clara, CA, USA). RNA libraries were constructed and sequenced using the Illumina HiSeq 2500 platform at the Centre for Applied Genomics, SickKids Hospital (Toronto, Canada), under a fee-for-service agreement. Differential gene expression analysis between MsDAD1-OE lines and wild-type plants was performed using the DESeq2 R package (version 1.20.0). P-values were adjusted for multiple testing, and genes with an adjusted p-value< 0.001 were defined as differentially expressed genes (DEGs). Gene Ontology (GO) enrichment analysis was performed, and GO terms with a corrected p-value < 0.05 were considered significantly enriched. Functional annotation of DEGs was carried out using the NR, GO, and KEGG databases.

All experiments and gene expression analyses were conducted with at least three independent biological replicates. Results are presented as mean values ± standard error (SE). Statistical analyses were performed using one-way analysis of variance (ANOVA). Asterisks above columns indicate statistically significant differences compared to the control: p < 0.05 (*) and p < 0.01 (**). Different letters above histogram bars denote significant differences among treatments at p < 0.05, as determined by post hoc multiple comparison tests.

Based on transcriptomic analysis of alfalfa (Medicago sativa L.) from our previous study (Dong et al., 2018), a gene encoding defender against apoptotic death 1 (DAD1), designated MsDAD1 was identified. Phylogenetic analysis revealed that this gene shares the highest sequence homology with MtDAD1 from Medicago truncatula, a model legume species, and was thus named MsDAD1 ( Figure 1A ). Expression analysis showed that MsDAD1 is significantly upregulated in response to heat stress ( Figure 1B ), suggesting its potential role in high-temperature adaptation. The coding sequence (CDS) of MsDAD1 was subsequently cloned using primers listed in Supplementary Table S1 . Consistent with other DAD1 orthologs, MsDAD1 encodes a protein with three predicted transmembrane (TM) domains (TM I/II/III) and contains a conserved oligosaccharyltransferase (OST) subunit domain ( Figure 1C ). Phylogenetic analysis further demonstrated that MsDAD1 shares 98%, 92%, and 87% sequence identity with DAD1 orthologs from M. truncatula, soybean (Glycine max), rice (Oryza sativa), and Arabidopsis thaliana, respectively, indicating that DAD1 is highly conserved across plant species ( Figure 1C ; Supplementary Table S1 ).

To investigate the role of MsDAD1 in heat stress tolerance, transgenic alfalfa plants constitutively expressing MsDAD1 were developed through stable genetic transformation. qRT-PCR analysis of six independent transgenic lines revealed that MsDAD1-OE1 and MsDAD1-OE3 exhibited the highest transcript levels and were selected for subsequent experiments ( Supplementary Figure S1 ). Phenotypic evaluation under heat stress showed that MsDAD1-OE plants were more resilient than wild-type (WT) plants. While WT plants displayed pronounced leaf chlorosis and abscission, MsDAD1-OE lines maintained greener, healthier foliage with reduced visible damage ( Figures 2A, B ).

Physiological responses of WT and MsDAD1-OE lines were further evaluated under both normal and heat stress conditions. Under non-stress conditions, no significant differences in relative water content (RWC) were observed between the genotypes ( Figure 2C ). However, upon exposure to heat stress, RWC declined in all plants, with MsDAD1-OE lines maintaining significantly higher RWC compared to WT ( Figure 2C ). Electrolyte leakage, assessed via ion conductivity, was significantly elevated in both genotypes under heat stress, but the increase was more pronounced in WT plants, indicating greater membrane damage ( Figure 2D ). Correspondingly, MsDAD1-OE lines retained higher chlorophyll content than WT under heat stress, consistent with delayed senescence phenotypes ( Figure 2E ).

As heat stress disrupts reactive oxygen species (ROS) homeostasis, the accumulation of hydrogen peroxide (H₂O₂), superoxide anion (O₂ ^-), and malondialdehyde (MDA) were quantified. Under heat stress, MsDAD1-OE lines exhibited significantly lower levels of H₂O₂ and O₂ ^- compared to WT ( Figures 2F, G ). MDA content, a marker of lipid peroxidation and oxidative damage, was also markedly reduced in MsDAD1-OE plants relative to WT ( Figure 2H ). These results suggest that MsDAD1 overexpression mitigates heat-induced oxidative damage by enhancing ROS scavenging capacity.

Collectively, these findings demonstrate that MsDAD1-OE lines outperform WT under heat stress, exhibiting enhanced physiological stability, reduced oxidative damage, and improved stress tolerance.

To elucidate the transcriptional changes regulated by MsDAD1, RNA-seq analysis on wild-type and MsDAD1-overexpressing (OE) alfalfa plants was performed. After quality control filtering, high-quality reads were retained for downstream analysis. A total of 1,088 differentially expressed genes (DEGs) were identified between WT and MsDAD1-OE lines, using thresholds of |Log₂ ^FoldChange| ≥ 1 and adjusted p-value < 0.05 ( Figure 3A ). Among these, 611 genes were upregulated and 477 genes were downregulated in MsDAD1-OE plants. qRT-PCR validation of six randomly selected genes confirmed the RNA-seq results, showing a high degree of consistency ( Figures 3B–G ).

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that DEGs were primarily associated with cellular and metabolic processes, biosynthesis of secondary metabolites, plant–pathogen interactions, MAPK signaling, plant hormone signal transduction, glutathione metabolism, and flavonoid and phenylpropanoid biosynthesis ( Figures 3H, I ; Supplementary Tables S2 , S3 ).

Given the critical role of plant hormones in heat stress responses, we focused on hormone-related genes. Transcriptome data revealed that MsDAD1-OE plants exhibited reduced expression of key genes involved in jasmonic acid (JA) and abscisic acid (ABA) biosynthesis specifically, LIPOXYGENASE1 (LOX1) and 9-cis-EPOXYCAROTENOID DIOXYGENASE1 (NCED1), respectively. This suggests that MsDAD1 may modulate JA and ABA metabolism during heat stress. To validate this, the expression of LOX1 and NCED1 in WT and MsDAD1-OE plants were examined under control and heat stress conditions. Under normal conditions, both genes showed slightly lower expression in MsDAD1-OE plants compared to WT, but the differences were not statistically significant (p >0.05). Upon exposure to high temperatures, expression of both LOX1 and NCED1 was significantly upregulated in both genotypes; however, the induction was notably stronger in WT plants ( Figures 4B–E, G ). LOX1 encodes a key enzyme in the JA biosynthetic pathway, catalyzing the conversion of α-linolenic acid to 13-hydroperoxyoctadecatrienoic acid (13-HPOT), a critical initial step in JA production ( Figure 4A ). NCED1 is the rate-limiting enzyme in ABA biosynthesis, catalyzing the oxidative cleavage of carotenoids to produce xanthoxin, a precursor of ABA ( Figure 4F ). To determine whether altered gene expression translated into changes in hormone levels, endogenous JA and ABA concentrations under both control and heat stress conditions were quantified. Heat stress significantly increased the accumulation of both hormones in WT and MsDAD1-OE plants; however, the increase was significantly greater in WT plants ( Figures 4I, J ).

Together, these results indicate that MsDAD1 negatively regulates the heat-induced accumulation of JA and ABA in alfalfa, likely through suppression of LOX1 and NCED1, contributing to enhanced stress tolerance.

To assess the involvement of JA and ABA in regulating leaf senescence in alfalfa, senescence phenotypes were evaluated following exogenous hormone treatments. Compared to the control, application of either JA or ABA significantly accelerated chlorophyll degradation, leaf yellowing, electrolyte leakage, and malondialdehyde (MDA) accumulation in both wild-type and MsDAD1-OE plants ( Figures 5A-D ). However, the severity of these senescence-associated responses was moderately attenuated in the MsDAD1-OE lines relative to wild-type plants. Under JA and ABA treatments, MsDAD1-OE plants retained higher chlorophyll content and exhibited lower electrolyte leakage and MDA levels compared to the wild type ( Figures 5B-D ). These findings suggest that MsDAD1 delays the progression of leaf senescence by limiting excessive JA and ABA accumulation, particularly under heat stress conditions.

Extensive evidence indicates that heat stress can significantly disrupt the vegetative-to-reproductive transition in plants. In this study, longitudinal monitoring of developmental progression revealed a marked delay in flowering time in MsDAD1-OE lines compared to wild-type (WT) plants ( Figure 6A ). Under normal growth conditions, MsDAD1-OE plants exhibited delayed flowering, as reflected by a longer time to floral initiation, increased node number, and greater plant height at flowering onset ( Figures 6B–D ). Although high-temperature stress accelerated flowering in alfalfa, this effect was notably attenuated in MsDAD1-OE lines, suggesting a role for MsDAD1 in modulating temperature-dependent flowering responses ( Figures 6B–D ).

Given the late-flowering phenotype of the MsDAD1-OE plants, the expression of flowering-related differentially expressed genes (DEGs) identified in our RNA-seq dataset were examined. Several key flowering regulators—such as FLOWERING LOCUS T (FT), EARLY FLOWERING 4 (ELF4), and FLOWERING TIME CONTROL GENE (FY)—were downregulated in MsDAD1-OE plants ( Figure 6E ). These genes are known to play critical roles in regulating flowering under heat stress conditions.

To validate these observations, we analyzed the expression of FT and ELF4 via qRT-PCR in both WT and MsDAD1-OE plants. Under normal conditions, expression levels of both genes were significantly lower in MsDAD1-OE lines compared to WT. Heat stress induced the expression of FT and ELF4 in both genotypes, but the induction was significantly stronger in WT plants ( Figures 6F, G ). These results suggest that MsDAD1 overexpression suppresses the heat-induced upregulation of flowering-promoting genes, thereby contributing to delayed flowering under elevated temperatures.