Effects of Exogenous Melatonin Concentration on Salt Stress Alleviation in Salix matsudana × S. alba Assessed Through Integrated Physiological Evaluation and Transcriptomic Analysis

Salt stress limits the establishment of Salix matsudana × S. alba seedlings. Exogenous melatonin (MT) can alleviate plant salt stress, but its concentration-dependent effects in Salix matsudana × S. alba remain insufficiently characterized. In this study, physiological responses were assessed across three NaCl levels and five MT concentrations, followed by transcriptome analysis of representative treatments. Under 6‰ NaCl, 0.5 mmol L−1 MT significantly reduced relative electrolyte conductivity and malondialdehyde content across three sampling times and increased total chlorophyll, proline, and soluble protein contents and antioxidant enzyme activities at the final sampling relative to the salt-only treatment. An integrated physiological assessment based on principal component analysis and supported by correlation analysis ranked 0.5 mmol L−1 highest under 6‰ NaCl, with 0.3 mmol L−1 ranking second, whereas 1.0 mmol L−1 generally provided no further overall improvement. Compared with the untreated control, the 6‰ NaCl plus 0.5 mmol L−1 MT treatment showed transcriptional changes involving plant hormone signal transduction and starch and sucrose metabolism. Quantitative reverse transcription PCR broadly supported RNA sequencing trends for nine candidate genes. These findings demonstrate concentration-dependent physiological protection and identify candidate pathways for investigating MT-associated salt stress alleviation in hybrid willow.