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Aerenchyma formation plays an important role in the survival of Potamogeton perfoliatus in submerged environment. To understand the regulatory role of reactive oxygen species (ROS) and caspase 3-like protein signaling molecules in aerenchyma formation, we investigated the effects of exogenous NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), catalase inhibitor (3-amino-1,2,4-triazole, AT), and caspase-3-like protein inhibitor (AC-DEVD-CHO, DEVD) on morphological and physiological characteristics and aerenchyma formation in P. perfoliatus. The results showed that after DPI treatment, caspase-3-like protein activity decreased, ROS-related enzyme activities increased, and H2O2 content decreased, thereby inhibiting aerenchyma formation. When the concentration of DPI was approximately 1 μmol/L, the inhibitory effect was the most obvious. On the contrary, after the AT treatment, caspase-3-like protein activity increased, ROS-related enzyme activities decreased, and the H2O2 content increased, ultimately promoting aerenchyma formation, and the promotion was the most obvious under treatment with approximately 500 μmol/L AT. After DEVD treatment, the inhibition of vegetative growth caused by DPI or AT treatment was alleviated, significantly reducing caspase-3-like activity and inhibiting aerenchyma development. The results of this study show that ROS has a positive regulatory effect on aerenchyma formation, and caspase-3-like protein is activated to promote ROS-mediated aerenchyma formation. This experiment provides a new theoretical basis for further exploration of the signal transduction effects of ROS and caspase-3-like protein in plant cells and their roles in plant development. [ABSTRACT FROM AUTHOR]