Functional Characterization of AfBBX from Amorpha fruticosa in Enhancing Osmotic and Salt-Alkali Tolerance in...

Drought and soil salinization severely limit the productivity of global agriculture and forestry, highlighting the urgency of identifying stress-resistant genes for molecular breeding. B-box (BBX) proteins constitute a class of zinc finger transcription factors that play significant roles in plant abiotic stress responses. Amorpha fruticosa (A. fruticosa) is a perennial woody plant with exceptional adaptability to harsh environments, serving as a valuable resource for mining stress-resistant genes. In this study, the AfBBX gene was cloned from A. fruticosa, and its function in stress tolerance was systematically analyzed. Bioinformatics analysis confirmed that AfBBX contains a conserved ZnF-BBOX domain and shares functional conservation with the BBX protein family. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed tissue-specific expression of AfBBX, with the highest expression in stems and the lowest in young leaves. Furthermore, AfBBX expression was dynamically regulated in roots and leaves of A. fruticosa under treatments of 5 μM ABA (drought mimic), H₂O₂ (oxidative stress), 10% PEG600 (osmotic stress), and NaHCO₃ (alkaline stress). Transgenic tobacco lines overexpressing AfBBX showed enhanced tolerance to osmotic and salt-alkali stresses at both germination and seedling stages. Meanwhile, compared to wild-type (WT) tobacco, transgenic lines exhibited higher germination rates, longer root lengths, and greater fresh weights under stress conditions. Under natural drought and salt-alkali stresses, transgenic tobacco maintained higher chlorophyll fluorescence intensity (Fv/Fm values), elevated activities of antioxidant enzymes [superoxide dismutase (SOD)], and reduced malondialdehyde (MDA) content. In conclusion, AfBBX enhances stress tolerance by mitigating photosystem damage, increasing reactive oxygen species (ROS) scavenging capacity, and reducing membrane lipid peroxidation. The findings from this study provide novel insights into the molecular mechanism underlying AfBBX-mediated stress resistance and offer valuable genetic resources for breeding drought- and salt-tolerant crops and forest trees.