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Beneficial root-associated fungi, particularly dark septate endophytes (DSEs), have emerged as potential modulators of stress tolerance, yet their underlying mechanisms in perennial fruit trees remain poorly understood. In this study, we investigated the effects of the DSE fungus Helotiales sp. S16 on drought responses of sweet cherry rootstock Gisela 5 seedlings. Fungal symbiosis was established by homogenizing fungal suspension with sterile growth substrate, and drought stress treatment was implemented 4\u00a0weeks after inoculation. Under drought conditions, inoculated seedlings accumulated markedly higher soluble sugar contents, which coincided with upregulated expression of carbohydrate-metabolism-related genes. Lipidomic and transcriptomic data demonstrated that fungal inoculation activated fatty-acid biosynthetic pathways and reshaped overall fatty-acid profiles. Hormone profiling showed elevated abscisic acid, jasmonic acid and salicylic acid levels, accompanied by suppression of auxin (IAA), and cytokinins (6-BA), indicating a reprogramming of hormonal crosstalk. Co-expression network analysis further identified the transcription factor PaHB12 as a regulatory hub in the S16-mediated drought response. These findings demonstrate that DSE fungus S16 enhances drought tolerance through integrated reprogramming of carbon allocation, membrane lipid composition and hormone crosstalk, providing a mechanistic basis for its potential application in sustainable orchard management.<\/jats:p>","DOI":"10.1093\/treephys\/tpag131","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T11:48:48Z","timestamp":1789040928000},"source":"Crossref","is-referenced-by-count":0,"title":["The dark septate endophyte S16 enhances drought tolerance in sweet cherry by coordinating metabolic reprogramming and the transcription factor PaHB12"],"prefix":"10.1093","volume":"46","author":[{"given":"Wenfei","family":"Li","sequence":"first","affiliation":[{"name":"College of Horticulture, Ludong University , 186 Hongqi Middle Road, Zhifu District, Yantai City, Shandong Province 264025 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