Genome-Wide Identification of Pineapple AcINH Genes and Functional Characterization of AcINH3 in Sucrose Metabolism and Drought Tolerance

Seasonal drought constitutes a major abiotic stress limiting the growth and yield of pineapple, a globally important Crassulacean acid metabolism (CAM) crop. The sucrose catabolism mediated by cell wall invertase (CWIN) plays a vital role in regulating plant growth and development, as well as adaptive responses to abiotic stresses. Invertase inhibitors (INHs) serve as specific post-translational regulators that modulate CWIN enzymatic activity. However, the INH family has not been systematically characterized in pineapple, and its functional roles in mediating sucrose metabolism and drought resistance remain elusive. In this study, three AcINHs were identified from the pineapple genome, followed by comprehensive analyses of their gene structures, phylogenetic relationships, homology characteristics and protein structures. Structural analysis revealed that all AcINH members harbor conserved motifs 1, 2, 3, 5 and 9, whereas only AcINH3 possesses motif 7. Expression analysis showed that only AcINH3 was significantly transcriptionally induced by drought stress among all family members. Functional validation demonstrated that AcINH3 knockout markedly elevated CWIN activity in pineapple seedling leaves, facilitating hexose accumulation and promoting plant growth and development. Moreover, AcINH3-edited lines exhibited enhanced drought resistance, accompanied by increased accumulation of soluble sugars (sucrose, glucose, fructose), abscisic acid (ABA), and proline (PRO), reduced malondialdehyde (MDA) content, and enhanced peroxidase (POD) activity. Biochemical assays further verified a direct physical interaction between AcINH3 and AcCWIN1, which mediates sucrose metabolism and drought stress responses. Collectively, this study identifies a novel AcINH3–AcCWIN1 post-translational module that modulates sugar metabolism and drought tolerance in pineapple, providing critical mechanistic insights for CAM plants. Our findings highlight AcINH3 as a promising target for genome-editing breeding to enhance drought resistance in CAM crops.