Nitric Oxide Functions as a Key Mediator in Brassinosteroid-Enhanced Alkaline Tolerance in Cucumber

This study investigated how exogenous 2,4-epibrassinolide (EBR) and nitric oxide (NO) enhance the tolerance of cucumber (<i>Cucumis sativus</i> L.) seedlings to NaHCO₃-induced alkaline stress under hydroponic conditions. NaHCO₃ exposure caused severe sodium toxicity, reactive oxygen species (ROS) accumulation, and photosynthetic inhibition, which, together, suppressed plant growth. Treatments with either EBR or NO significantly improved plant performance by alleviating these adverse effects. Both regulators enhanced the ROS scavenging system, maintained ionic homeostasis, and alleviated sodium toxicity. They also stimulated the activities of vacuolar H⁺-ATPase, H⁺-PPase, and plasma membrane H⁺-ATPase, and increased the accumulation of citric and malic acids, thereby sustaining higher photosynthetic efficiency under stress conditions. qRT-PCR analysis further revealed that EBR and NO upregulated <i>SOS1</i> and <i>NHX2</i> (sodium transporters) as well as <i>PIP1;2</i> and <i>PIP2;4</i> (aquaporins), confirming their involvement in ionic and osmotic regulation. Pharmacological experiments showed that application of NO synthesis inhibitors, including tungstate and L-NAME, as well as the NO scavenger cPTIO, markedly weakened the protective effects of EBR. In contrast, application of the brassinosteroid biosynthesis inhibitor brassinazole (BRz) only had a limited effect on NO-mediated stress tolerance. Collectively, these findings demonstrate that NO functions as a downstream signaling mediator of EBR, coordinating multiple defense pathways including photosynthetic regulation, antioxidant protection, ion balance, aquaporin activity, and organic acid metabolism to enhance cucumber resistance to alkaline stress.