Hydrogen sulfide pretreatment mitigates the adverse effects of salinity in young carob seedlings

Despite the inherent high salinity tolerance of mature carob (Ceratonia siliqua L., Fabaceae) trees, young seedlings are particularly vulnerable to salt stress, which can hinder their growth and development. Although hydrogen sulfide (H₂S) has been widely studied as a regulator of salinity tolerance in herbaceous plants, its role in woody legumes remains largely unexplored. This study aimed to explore the potential of hydrogen sulfide (H₂S) pretreatment to enhance the salinity tolerance of young carob seedlings. A pot experiment was conducted using a factorial design to evaluate the effects of sodium hydrosulfide (NaHS, an H₂S donor) at 0, 75, and 200 µM under saline conditions (0, 50, and 100 mM NaCl). The results indicated that H₂S pretreatment enhanced the biomass of both shoots and roots under both saline and non-saline conditions, with the potential to compensate growth loss due to salt stress. Additionally, stress markers such as hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) increased by about 23% and 28%, respectively, in seedlings treated with 75 µM NaHS after exposure to 100 mM NaCl stress. The enhanced salinity stress tolerance was presumably associated with increased antioxidant enzyme activity, including superoxide dismutase (SOD, ≈ 24%), ascorbate peroxidase (APX, ≈ 118%), and peroxidase (POD, ≈ 50%) after 75 µM NaHS pretreatment in plants exposed to 100 mM NaCl. Moreover, NaHS pretreatment reduced sodium ion (Na⁺) accumulation in both leaves and roots. These findings indicate that H₂S enhances the salinity tolerance of carob seedlings, helping them better withstand suboptimal growing conditions.