Insights into α-amylase, invertase, and amyloglucosidase functions in resistant wheat cultivar carbohydrate osmoprotectants production in response to sorbitol and mannitol-induced osmotic stress

Abstract Studying wheat seed germination and seedling development under osmotic stress could improve our understanding of wheat adaptation to drought. This study used PEG, sorbitol, and mannitol to elucidate the effects of osmotic stress on germinating seeds of the Tsholoha wheat cultivar. In vitro activity assays were conducted to elucidate inhibitory effects of PEG, sorbitol, and mannitol on β-glucosidase, invertase (INV), and amyloglucosidase (AMG) activity; followed by molecular docking analysis of sorbitol and mannitol affinity binding to α-amylase and AMG. Results showed that germination percentage, roots, and coleoptiles decreased in PEG-, sorbitol-, and mannitol-treated samples. At 10% and 20% sorbitol and mannitol treatments affected seed morphological parameters more than the PEG treatments. The treatments led to more than 50% higher starch and sucrose accumulation relative to the control. In addition, amylase activity was reduced by more than 30%, followed by 90% reduction of INV and AMG activity. The in vitro activity findings confirmed that PEG, sorbitol, and mannitol inhibit these enzymes. The kinetic analysis showed sorbitol inhibited the molecules through uncompetitive inhibition, whereas PEG and mannitol used a mixed inhibition mechanism. Molecular docking simulations confirmed that mannitol and sorbitol bind to the amylase and AMG active sites, leading to competitive inhibition. This study demonstrates that inhibition of α-amylase, INV, and AMG led to the accumulation of oligosaccharide osmoprotectants (starch, maltooligosaccharides, sucrose, and trehalose) and proline, which protect wheat seeds germinating under osmotic stress conditions.