Effect of exogenous melatonin on physiological characteristics of wild tetraploid strawberry under saltalkali stress and preliminary screening of salt-alkali tolerant candidate genes

【Objective】The soil and substrate salinization problem in China's strawberry cultivation is becoming increasingly severe, hindering the sustainable development of the strawberry industry; meanwhile, China possesses abundant wild strawberry resources whose resistance genes, evolved over millions of years, remain worthy of further exploration and utilization. This study aimed to investigate the physiological effects of exogenous melatonin on wild tetraploid strawberries under saline–alkali stress conditions, as well as the changes in transcriptional expression of related genes, and to preliminarily screen candidate genes associated with salt–alkali tolerance.【Methods】Thirty-six wild tetraploid strawberry accessions were systematically evaluated for salt- alkali tolerance. Plants were assigned to three treatments: water control (CK), salt-alkali treatment (SA), and salt-alkali treatment supplemented with melatonin (SA+MT). After treatment, we measured growth- related traits and calculated the salt injury index to comprehensively assess the salt-alkali tolerance of each accession. Based on the screening results, salt-alkali tolerant and highly salt-alkali sensitive accessions were selected for further physiologi-cal and molecular analysis. We then determined plant phenotypes and antioxidant enzyme activities, and performed transcriptome sequencing to identify differentially expressed genes (DEGs). These DEGs were further analyzed via GO functional enrichment, KEGG pathway enrichment, weighted gene co- expression network analysis (WGCNA), and protein- protein interaction (PPI) network analysis to screen for candidate genes closely associated with plant responses to salt-alkali stress and melatonin-mediated regulation. The 2-kb upstream promoter regions of the candidate genes were further analyzed to identify cis-acting regulatory elements, and their encoded proteins were subjected to predicted subcellular localization. Finally, the expression patterns of the candidate genes were validated via qRT- PCR. 【Results】Phenotypic evaluation after salt-alkali stress identified two salt-alkali tolerant accessions and two highly salt-alkali sensitive accessions. Exogenous melatonin significantly alleviated salt-alkali-induced growth inhibition in the sensitive accessions, leading to a clear recovery in plant height, root length, and fresh weight. Compared with treatment with salt-alkali stress alone, melatonin application significantly increased the activities of SOD, POD, and CAT, while reducing MDA accumulation. This indicates that exogenous melatonin alleviates oxidative damage by enhancing ROS scavenging under salt- alkali stress. KEGG enrichment analysis revealed that the MAPK signaling pathway was significantly enriched after exogenous melatonin treatment under salt-alkali stress, indicating that MAPK-mediated signal transduction may be involved in the melatonin- regulated response to salt- alkali stress. WGCNA identified multiple gene modules significantly associated with salt-alkali stress and melatonin treatment. By integrating WGCNA with GO enrichment analysis, we identified 78 salt stress- related genes in total, which include multiple members from the mitogen-activated protein kinase (MAPK) signaling pathway and the calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CBL-CIPK) signaling pathway. Furthermore, protein- protein interaction (PPI) network analysis revealed potential crosstalk between the MAPK and CBL-CIPK signaling pathways mediated by the reactive oxygen species (ROS)-associated protein RBOHF and the calcium-binding protein CP1. In addition, multiple CIPK proteins were predicted to interact with the Stelar K+ Outward Rectifier (SKOR) potassium channel, suggesting that coordinated regulation of ROS homeostasis and potassium transport by the MAPK and CBL-CIPK signaling networks may contribute to the adaptation of wild tetraploid strawberry to salt-alkali stress. Based on the integrated results, we identified three candidate genes associated with salt-alkali tolerance: the FmMPK7, FmMKK9-A, and FmMKK9-B. Promoter analysis showed that the 2-kb upstream regions of all three genes contain abundant cis- acting elements associated with transcriptional regulation, hormone signaling, and abiotic stress responses. Among them, the FmMPK7 was significantly upregulated under salt-alkali stress, while the FmMKK9-A and FmMKK9-B were markedly induced after melatonin treatment.【Conclusion】Exogenous melatonin treatment can alleviate the growth inhibition of strawberry caused by salt-alkali stress.