Effect of MT on the growth of NaCl-stressed alfalfa plants
To study the seed germination and growth under salt stress, we counted the germination rate of alfalfa sprouts and measured their root length and fresh weight. Analysis of germination rates over seven days demonstrated differences among the treatments (Fig 1A). On the seventh day, seeds treated with 200 μM and 300 μM MT exhibited higher germination rates compared to the salt stress group without MT. Among, the 300 μM MT treatment resulted in germination rates closest to the CK group (Fig 1B). In terms of fresh weight and root length, comparison between the salt stress groups treated with various concentrations of MT and the CK group revealed interesting results. The CK group had a fresh weight of 0.2553 g and a root length of 5.82 cm. The group treated with 10 μM MT closely resembled the CK group, with a fresh weight of 0.2053 g and a root length of 3.33 cm. In contrast, the salt stress group without MT exhibited a fresh weight of 0.1884 g and a root length of 3.02 cm (Fig 1C, 1D). These findings suggest the efficacy of 10 μM MT in ameliorating the growth of salt-stressed plants, as evidenced by superior fresh weight and root length outcomes compared to other MT concentrations.The observed improvement in germination and growth parameters under MT treatment aligns with previous studies showing that exogenous melatonin enhances plant tolerance to abiotic stresses by promoting growth and mitigating stress-induced damage
(Chen et al., 2018a; Zhang et al., 2021). This indicates that melatonin may act as a growth regulator to counteract the inhibitory effects of salt stress on alfalfa seed germination and seedling development.
Changes of indicators in oxidation system
To investigate the mechanism by which melatonin enhances alfalfa salt tolerance, we analyzed oxidative stress indices under various treatments. Our examination of different concentrations of MT in alfalfa growth conditions under salt stress revealed that the group treated with 10 μM MT under salt stress exhibited the most favorable outcomes. Consequently, we assessed the physiological indexes in the control group (CK), the 200 mM NaCl treatment group and the group treated with 10 μM MT under NaCl stress. It was found that SOD enzyme activity was reduced in the salt-stressed group, while POD, MDA, O2-• and GSH contents were increased in the salt-stressed group compared to the CK group. Interestingly, when 10 μM MT was added to the salt stress group, the activity of POD increased significantly by 10.17%, while the levels of MDA, O2-• and GSH decreased by 14.91%, 18.22% and 18.95%, respectively, with minimal change in SOD activity (Fig 2A-2E). These results indicate that melatonin alleviates salt-induced oxidative damage by modulating the antioxidant system. Under salt stress, plants accumulate reactive oxygen species (ROS) such as O2-•, leading to lipid peroxidation (reflected by elevated MDA levels) and oxidative stress
(Tyerman et al., 2019). The melatonin-enhanced POD activity and reduced ROS accumulation align with previous findings that melatonin activates antioxidant enzymes to scavenge excess ROS
(ElSayed et al., 2021). Furthermore, the decrease in GSH levels in MT-treated groups may reflect its utilization in ROS detoxification, as GSH is a key antioxidant involved in maintaining redox homeostasis
(Gill et al., 2013). Collectively, these findings suggest that melatonin improves salt tolerance by enhancing the antioxidant capacity of alfalfa seedlings.
Differential expression analysis and cluster analysis of genes in alfalfa seedlings
Transcriptomic analysis of WLCK, WLN and WLNMT identified 3,547 differentially expressed genes (DEGs). Compared to WLCK, WLN had 726 up-regulated and 1,405 down-regulated genes, while WLNMT had 1,097 up-regulated and 1,799 down-regulated genes. Only 31 up-regulated and 58 down-regulated genes were found in WLNMT vs. WLN (Fig 3A). A Venn diagram showed minimal overlap (0.14%) among all three groups, with most DEGs being unique to WLNMT vs. WLCK (37.61%) (Fig 3B). ANOVA and heatmap clustering confirmed significant expression differences across treatments (Fig 3C, 3D).The large number of DEGs in WLNMT vs. WLCK suggests melatonin modulates a broad range of genes under salt stress. This aligns with studies showing melatonin regulates stress-responsive gene expression by interacting with signaling pathways or hormones
(Gou et al., 2018). The limited DEGs between WLNMT and WLN indicate melatonin primarily modulates genes already affected by salt stress, rather than activating entirely new pathways, supporting its role in enhancing existing stress responses.
GO annotation analysis and KEGG enrichment pathway analysis of DEGs
GO annotation revealed DEGs in all comparisons were enriched in biological processes (metabolic processes, cellular processes), cellular components (cell parts, membrane parts) and molecular functions (binding, catalytic activity), with stronger enrichment in WLNMT vs. WLCK (Fig 4A). KEGG analysis showed that DEGs of WLN vs. WLCK were enriched in porphyrin metabolism and linoleic acid metabolism pathways; The DEGs of WLNMT vs. WLCK are enriched in the biosynthesis pathways of isoflavones and alkaloids; The DEGs of WLNMT vs. WLN are enriched in ABC transporters and photosynthetic pathways (Fig 4B-4D). The enrichment of metabolic and cellular processes in GO terms suggests salt stress and melatonin primarily affect basic physiological functions. KEGG pathways related to secondary metabolism (
e.
g., isoflavones, flavonoids) and transporters (ABC) highlight key mechanisms. Isoflavonoids and flavonoids are known to enhance antioxidant capacity and stress tolerance (
Back, 2021), while ABC transporters mediate the transport of stress-related compounds
(Kang et al., 2011). The stronger enrichment in WLNMT vs. WLCK indicates melatonin amplifies these pathways to improve salt tolerance.
Gene co-expression networks
WGCNA identified 13 co-expression modules. The turquoise module (4,804 DEGs) correlated positively with germination rate, root length, fresh weight and SOD (correlation coefficients 0.683-0.954), while genes in the blue module (3,676 DEGs) exhibited positive correlations with POD, GSH, MDA and O2-• (0.633-0.765) (Fig 5A, 5B). Genes in these modules were associated with flavonoid biosynthesis, glutathione metabolism, phytohormone signaling, MAPK pathways and ABC transporters (Fig 5C, 5D).These modules link gene expression with physiological characteristics. The turquoise module likely contributes to growth recovery under MT treatment, while the blue module is involved in oxidative stress responses. The association with flavonoid and glutathione pathways supports their roles in ROS scavenging
(Gill et al., 2013; Back, 2021). MAPK signaling and phytohormone pathways further indicate melatonin regulates stress signaling networks
(Kumar et al., 2020; Waadt et al., 2022).
Transcriptome analysis
Transcriptomic analysis revealed up-regulation of flavonoid biosynthesis genes (HCT, DFR, CYP81E9) in WLN vs. WLCK and WLNMT vs. WLCK. Among them, the MT-treated groups showed greater up-regulation than salt-only groups. Flavonoids are critical antioxidants and their accumulation enhances salt tolerance by scavenging ROS (
Back, 2021). DFR up-regulation stimulates anthocyanin synthesis, which improves stress tolerance in crops like rapeseed
(Kim et al., 2017). Melatonin’s promotion of these genes suggests it enhances flavonoid synthesis to mitigate salt stress damage.
Genes involved in glutathione metabolism (speE, DHAR, GST, APX) were up-regulated in WLN vs. WLCK and more so in WLNMT vs. WLCK. DHAR enhances GSSG-to-GSH conversion, while GST accelerates glutathione-mediated detoxification
(Reinemer et al., 1992; Chen et al., 2022). Glutathione is central to ROS scavenging
(Gill et al., 2013). Up-regulation of these genes under MT treatment indicates enhanced glutathione recycling and detoxification, reducing oxidative damage. This aligns with physiological data showing lower MDA and O2-• in MT-treated plants, confirming glutathione’s role in melatonin-mediated salt tolerance.
This study confirms the potential of melatonin to enhance salt tolerance in alfalfa under controlled conditions. However, its efficacy may be influenced by variations in temperature, light and soil properties and these findings may not extrapolate directly to field environments. Further validation through field trials is essential to assess melatonin’s effectiveness under real-world conditions, where complex interactions among environmental variables occur.
qRT-PCR validation
qRT-PCR analysis of eight selected genes showed mRNA relative abundance trends consistent with RNA-seq results (Fig 6), validating transcriptome reliability. These genes, involved in pathways like flavonoid biosynthesis and hormone signaling, likely play key roles in salt stress responses.