24-Epibrassinolide enhances aluminum tolerance in tobacco through Ca²⁺-dependent signaling, antioxidant regulation, and metal homeostasis

Aluminum (Al³⁺) toxicity is a major limitation to plant productivity in acidic soils, disrupting cellular homeostasis, redox balance, and nutrient uptake. Brassinosteroids are key regulators of plant stress signaling, yet their role in Al³⁺ tolerance remains insufficiently understood. Here, we investigated the signaling functions of 24-epibrassinolide (24-EBL) in mediating aluminum stress responses in <i>Nicotiana tabacum</i> grown under soilless culture conditions. Exogenous 24-EBL significantly alleviated Al³⁺-induced photosynthetic inhibition, as reflected by increased transpiration rate (Tr), stomatal conductance (Gs), net photosynthetic rate (Pn), electron transport rate (ETR), and effective quantum yield of PSII (ΦPSII). Enhanced non-photochemical quenching (NPQ) indicated improved dissipation of excess excitation energy, suggesting photoprotective regulation. At the molecular level, 24-EBL treatment upregulated the antioxidant defense genes CAT1, NtPOD1, and NtSOD3, leading to increased enzymatic activities and reduced reactive oxygen species (ROS) accumulation, thereby preserving membrane stability. Notably, 24-EBL modulated metal detoxification pathways by inducing the expression of the phytochelatin-related genes Pr8 and Pr2, along with Al-ATPase transporters associated with vacuolar sequestration. This was accompanied by altered ion homeostasis, where enhanced Ca²⁺ and K⁺ uptake antagonized Al³⁺ accumulation and restricted its translocation to shoots. The marked upregulation of calmodulin (CaM) suggests that Ca²⁺-dependent signaling plays a central role in 24-EBL-mediated aluminum tolerance. Correlation analysis revealed strong associations between CaM expression, photosynthetic efficiency, antioxidant capacity, and metal detoxification markers. Together, these findings indicate that 24-EBL enhances aluminum tolerance in tobacco through a coordinated signaling network involving Ca²⁺-mediated signal transduction, redox regulation, and metal homeostasis. This study highlights brassinosteroid-calcium crosstalk as a key regulatory module in plant adaptation to aluminum stress.