Integrated Physiological and Transcriptomic Analyses Elucidate the Molecular Mechanisms of Exogenous Melatonin-Mediated Salt Tolerance in Pomegranate (Punica granatum L.)

Yuanling YangHuiying LiuHaosheng GaiYang LiuMingxuan LiuLinnan Wu^*Ming Diao^*

• Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources Utilization of Xinjiang Production and Construction Corps, College of Agriculture, Shihezi University, Shihezi, China

Salt stress is a critical constraint affecting the cultivation of Tunisian soft-seeded pomegranate (Punica granatum L.). To elucidate the molecular mechanisms underlying exogenous melatonin (MT)-mediated enhancement of salt tolerance in pomegranate seedlings, this study integrated physiological phenotyping and transcriptome sequencing to systematically investigate MT's regulatory effects on antioxidant systems, photosynthetic apparatus function, osmotic adjustment, and core metabolic pathways under salt stress. The results demonstrated that 200 mM NaCl treatment induced reactive oxygen species (ROS) overaccumulation, elevating malondialdehyde (MDA) content and relative electrical conductivity (REC) by 0.43 and 0.46 fold, respectively. Concurrently, salt stress severely impaired photosynthetic performance: PSII maximum photochemical efficiency (FV/FM) decreased by 44.5%, actual photochemical efficiency (YII) and photochemical quenching (qP) were reduced, and non-photochemical quenching (NPQ) increased, indicating serious photoinhibition and energy wastage. In contrast, 400 μM MT treatment effectively mitigated oxidative damage by coordinated activation of superoxide dismutase (SOD, +14.3%), peroxidase (POD, +21.7%), and catalase (CAT, +11.7%) activities, thereby stabilising membrane integrity. Furthermore, MT significantly alleviated photoinhibition: FV/FM increased by 39%, YII and qP rose, and NPQ decreased compared to salt-stressed plants, reflecting enhanced protection of the PSII reaction center and optimized light energy allocation. Transcriptomic profiling revealed that MT optimised carbon allocation through upregulation of TPS (trehalose-6-phosphate synthase) for osmotic adjustment and sucrose metabolism genes (SUS, UGP2), while restoring starch biosynthesis via glgA expression. Weighted gene co-expression network analysis (WGCNA) identified two hub modules: the blue module, whcih was enriched in antioxidant-related genes (e.g., LOC116212144) and the yellow module, which was linked to membrane stabilization (e.g., LOC116203737). Furthermore, MT alleviated the transcriptional repression of the MAPKK kinase gene MKK9, thereby re-establishing ANP1-MKK9-MAPK signaling cascades to activate downstream defense gene PR1 and remodel phytohormone networks. This study provides the first evidence of a multi-tiered regulatory framework—spanning "antioxidant defense, photosynthetic protection, osmotic homeostasis, and This is a provisional file, not the final typeset article stress signaling"—through which MT enhances salt tolerance in pomegranate, offering actionable insights for breeding programs and cultivation practices in saline-alkaline regions.