Enhancing salt tolerance in soybeans through co-overexpression of bioactive peptides “Lunasin and Bowman-Birk Protease Inhibitor” and evaluating their antioxidant activity in vitro

Lunasin, a bioactive peptide originally discovered in soybeans, is protected by the Bowman-Birk protease inhibitor (BBI), preventing its digestion by pancreatin and allowing it to reach target tissues and organs in a biologically active state. Both Lunasin and BBI have demonstrated various chemopreventive functions such as anti-inflammatory, anticarcinogenic, and antioxidant properties. Previous research indicated that the overexpression of Lunasin and BBI enhances tolerance to salt and water deficit stresses in transgenic (TG) plants. This study investigated the combined effects of Lunasin and BBI overexpression in soybeans under salt stress conditions. The results showed that soybean plants co-overexpressing Lunasin and BBI significantly outperformed plants overexpressing either gene alone or wild-type (WT) plants, yielding 197% more seeds under stress. Additionally, the protein samples from the co-overexpressing TG line exhibited higher antioxidant activity compared to samples from single gene overexpression lines. These findings validate the hypothesis that stacking the overexpression of two genes can substantially enhance stress tolerance, offering a proof of concept for improving salt tolerance in various plants, including important crops. Furthermore, this discovery holds significant implications for genetically engineering crops to improve yield. Another objective of this study was to produce enriched Lunasin/BBI soybeans, which possess enhanced chemopreventive properties and could serve as valuable resources for functional food applications.