BRI1-EMS-SUPPRESSOR 1 (BES1) is a plant-specific transcription factor mediating brassinosteroid (BR) signaling and regulating cell proliferation, differentiation, morphogenesis, and stress responses. Tobacco ( Nicotiana tabacum ) is an important model plant and crop, but the BES1 family in tobacco remains poorly understood. In this study, we identified 17 BES1 genes and analyzed their tissue-specific expression and responses to hormones and abiotic stresses. NtBES1–17 was down-regulated by BR and up-regulated by brassinazole, indicating a key role in BR signaling, while NtBES1–1 and NtBES1–6 responded strongly to stress. Subcellular localization analysis showed that NtBES1–1 was localized in the nucleus, while NtBES1–6 and NtBES1–17 exhibited nucleo-cytoplasmic localization. These three proteins were capable of forming heterodimers and interacting with BIN2-like kinases. Overexpression of NtBES1–17 increased leaf and branch numbers, delayed flowering and senescence, and produced larger seeds, whereas knockout lines displayed opposite phenotypes. Hormone quantification and transcriptome analysis revealed increased feedback inhibition of BR biosynthesis and an antagonistic interaction with the jasmonic acid (JA) signaling pathway in the overexpression lines, while KEGG analysis showed that genes associated with ribosome, carbon fixation, and amino acid biosynthesis were widely down-regulated in the NtBES1–17/-11 knockout lines. Collectively, this study provides a systematic characterization of the BES1 family in tobacco and highlights NtBES1–17 as a central regulator of growth, development, and yield traits, offering molecular targets for crop improvement and stress resilience.
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