Ethylene-mediated CsWRKY41 regulates the PYL-PP2C module to promote adventitious root formation in cucumber under waterlogging stress

Waterlogging stress significantly compromises crop growth and yield. In cucumber (Cucumis sativus L.), the formation of adventitious roots (ARs) is a critical morphological adaptation for survival. While ethylene is known to promote AR formation under waterlogging, the interplay between ethylene and abscisic acid (ABA) signaling remains complex. In this study, we modulated ethylene levels using the precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the inhibitor aminoethoxyvinylglycine (AOA) to investigate the underlying transcriptional networks. RNA-seq analysis of hypocotyls under waterlogging (WL), WL+ACC, and WL+AOA conditions identified a set of ethylene-responsive genes, including the transcription factor CsWRKY41. Functional characterization revealed that CRISPR/Cas9-mediated mutation of CsWRKY41 significantly impaired AR formation, whereas its overexpression enhanced it. Mechanistically, CsWRKY41 directly binds to W-box elements within the promoters of the ABA receptor gene CsPYL4 and the phosphatase gene CsPP2C24, acting as a transcriptional activator of CsPYL4 and a repressor of CsPP2C24. Furthermore, CsPYL4 physically interacts with CsPP2C24 to modulate ABA signaling. Overexpression of CsPYL4 was found to upregulate the peroxidase gene CsPrx2 and increase H2O2 accumulation, thereby facilitating AR development. Collectively, our findings elucidate a novel molecular mechanism wherein ethylene orchestrates AR formation via the CsWRKY41-CsPYL4-CsPP2C24 module to mitigate waterlogging stress.