CmHRE2L-CmACS6 transcriptional cascade negatively regulates waterlogging tolerance in Chrysanthemum.

The role of ethylene as an initial signaling molecule in waterlogging stress is well-established. However, the complex molecular mechanisms underlying ethylene biosynthesis and its functional significance in chrysanthemums under waterlogging conditions have remained unclear. In this study, we observed an increase in the expression of 1-aminocyclopropane-1-carboxylate synthase 6 (CmACS6), which encodes a key enzyme responsible for ethylene biosynthesis, in response to waterlogging. This elevation increases ethylene production, induces leaf chlorosis, and enhances the chrysanthemum's sensitivity to waterlogging stress. Moreover, our analysis of upstream regulators revealed that the expression of CmACS6, in response to waterlogging, is directly upregulated by CmHRE2-like (Hypoxia Responsive ERF-like, CmHRE2L), an ethylene response factor. Notably, CmHRE2-L binds directly to the GCC-like motif in the promoter region of CmACS6. Genetic validation assays demonstrated that CmHRE2L was induced by waterlogging and contributed to ethylene production, consequently reducing waterlogging tolerance in a partially CmACS6-dependent manner. This study identified the regulatory module involving CmHRE2L and CmACS6, which governs ethylene biosynthesis in response to waterlogging stress.