Abstract

Glycine betaine (GB), a key osmoprotectant in plants, alleviates abiotic stress through osmotic adjustment and reactive oxygen species (ROS) scavenging. Although betaine aldehyde dehydrogenase (BADH) catalyzes GB biosynthesis and enhances salinity tolerance in crops such as rice and maize, its role in waterlogging adaptation remains unclear. A putative BADH gene, HfBADH1 , was cloned from waterlogging-treated daylily ( Hemerocallis spp.) ‘Autumn Red’. HfBADH1 expression increased 6.8-fold under waterlogging and 8.9-fold under salinity. Notably, HfBADH1 expression was repressed by abscisic acid (ABA), in contrast with typical stress-responsive genes. In addition, the activities of BADH and aldehyde dehydrogenase (ALDH) correlated with waterlogging tolerance among different daylily cultivars. Overexpression of HfBADH1 in Arabidopsis thaliana under the CaMV35S promoter resulted in 2.4-fold higher BADH activity and twofold higher ALDH activity than in wild-type plants. The transgenic lines exhibited enhanced tolerance to both salinity and waterlogging. Stress responses were evaluated by antioxidant and anaerobic respiration enzyme activities, and physiological indices including malondialdehyde (MDA) levels and chlorophyll content. The observed stress tolerance in transgenic A.thaliana suggested that BADH overexpression helps stabilize cellular structures and scavenges ROS. The increase in ALDH activity indicated that HfBADH1 overexpression may facilitate aldehyde oxidation, supporting adaptation to hypoxic conditions induced by waterlogging. This study is the first to link BADH overexpression to dual stress resilience, highlighting its combined enzymatic roles in GB biosynthesis and aldehyde detoxification. The ABA-independent regulatory pattern provides insight into noncanonical stress adaptation and suggests new strategies for engineering crops tolerant to saline and flood-prone environments.