Exogenous melatonin enhances salt tolerance by regulating the phenylpropanoid biosynthesis pathway in common bean at sprout stage

Abstract

Salt stress is a major environmental abiotic stress factor. Plants sense salt from germination onwards, negatively affecting their growth and development. Enhancing salt tolerance in crops at the sprout stage is crucial, given that it is the first stage to encounter stress. Melatonin (N-acetyl-5-methoxytryptamine) is a potent antioxidant that can alleviate stress from various environmental factors. Here, a common bean variety “Heiyundou” was used as the plant material. A concentration of 70 mMol·L⁻¹ NaCl was chosen as the stress treatment, and 100 μmol·L⁻¹ melatonin was applied. Four treatment groups were established: CK (control, water treatment), S (salt stress), M (melatonin), and M+S (salt stress with melatonin). Melatonin application under salt stress (M+S) significantly improved sprout length, surface area, volume, and average diameter compared to the salt stress group (S). Physiological analysis revealed that salt stress increased the activity of reactive oxygen species (ROS) scavenging enzymes, while exogenous melatonin (M+S) further enhanced this activity. Salt stress also significantly elevated levels of stress markers like malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂⁻). However, these markers decreased under the M+S treatment, indicating melatonin's protective effect. RNA sequencing (RNA-Seq) analysis identified 639 differentially expressed genes (DEGs) between the control (W) and salt stress (S) groups, and 170 DEGs between the salt stress (S) and salt stress with melatonin (M+S) groups. 40 DEGs were common to both comparisons (Co-DEGs). Gene Ontology (GO) enrichment analysis revealed that oxidoreductase activity (GO:0016491) and oxidation–reduction processes (GO:0055114) were enriched in all three groups (WvsS, SvsM+S, and Co-DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that phenylpropanoid biosynthesis (Ko00940) was the most enriched pathway in all three groups. Within this pathway, 4-coumarate-CoA ligase (4CL) and peroxidase (POD) were identified as key enzymes. Molecular docking simulations further confirmed the binding potential of melatonin to these two enzymes. Additionally, 4CL activity and lignin content analyses supported the role of phenylpropanoid biosynthesis as the underlying mechanism of melatonin's protective action. Collectively, these findings provide a theoretical basis for applying melatonin in enhancing salt tolerance in common bean crops.