| Summary: | As a brassinosteroid (BR), 24-epibrassinolide (24-epiBL) has been widely used to enhance the resistance of plants to multiple stresses, including salinity. Black locust (<i>Robinia pseudoacacia</i> L.) is a common species in degraded soils. In the current study, plants were pretreated with three levels of 24-epiBL (0.21, 0.62, or 1.04 µM) by either soaking seeds during the germination phase (Sew), foliar spraying (Spw), or root dipping (Diw) at the age of 6 months. The plants were exposed to salt stress (100 and 200 mM NaCl) via automatic drip-feeding (water content ~40%) for 45 days after each treatment. Increased salinity resulted in a decrease in net photosynthesis rate (<i>P</i><sub>n</sub>), stomatal conductance (<i>G</i><sub>s</sub>), intercellular:ambient CO<sub>2</sub> concentration ratio (<i>C</i><sub>i</sub>/<i>C</i><sub>a</sub>), water-use efficiency (<i>WUE</i><sub>i</sub>), and maximum quantum yield of photosystem II (PSII) (<i>F</i><sub>v</sub>/<i>F</i><sub>m</sub>). Non-photochemical quenching (<i>NPQ</i>) and thermal dissipation (<i>H</i><sub>d</sub>) were elevated under stress, which accompanied the reduction in the membrane steady index (MSI), water content (RWC), and pigment concentration (Chl a, Chl b, and Chl). Indicators of oxidative stress (i.e., malondialdehyde (MDA) and antioxidant enzymes (peroxidase (POD) and superoxide dismutase (SOD)) in leaves and Na<sup>+</sup> content in chloroplasts increased accompanied by a reduction in chloroplastid K<sup>+</sup> and Ca<sup>2+</sup>. At 200 mM NaCl, the chloroplast and thylakoid ultrastructures were severely disrupted. Exogenous 24-epiBL improved MSI, RWC, K<sup>+</sup>, and Ca<sup>2+</sup> content, reduced Na<sup>+</sup> levels, maintained chloroplast and thylakoid membrane structures, and enhanced the antioxidant ability in leaves. 24-epiBL also substantially alleviated stress-induced limitations of photosynthetic ability, reflected by elevated chlorophyll fluorescence, pigment levels, and <i>P</i><sub>n</sub>. The positive effects of alleviating salt stress in <i>R. pseudoacacia</i> seedlings in terms of treatment application was Diw > Sew > Spw, and the most positive impacts were seen with 1.04 µM 24-epiBL. These results provide diverse choice for 24-epiBL usage to defend against NaCl stress of a plant.
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