RAPID LEAF FALLING 1 facilitates chemical defoliation and mechanical harvesting in cotton.

Chemical defoliation stands as the ultimate tool in enabling the mechanical harvest of cotton, offering economic and environmental advantages. However, the underlying molecular mechanism that triggers leaf abscission through defoliant remains unsolved. In this study, through single-nucleus mRNA sequencing (snRNA-seq) of the abscission zone (AZ) from cotton petiole, we meticulously constructed a transcriptomic atlas and identified two newly-formed cell types, abscission cells and protection layer cells in cotton petiole AZ after defoliant treatment. GhRLF1 (RAPID LEAF FALLING 1), as one of the members encoding cytokinin oxidase/dehydrogenase (CKX) gene family, was identified as key marker gene unique to the abscission cells following defoliant treatment. Overexpression of GhRLF1 resulted in reduced cytokinin accumulation and accelerated leaf abscission. Conversely, CRISPR/Cas9-mediated loss of GhRLF1 function appeared to delay this process. Its interacting regulators, GhWRKY70, acting as "Pioneer" activator, and GhMYB108, acting as "Successor" activator, orchestrate a sequential modulation GhWRKY70/GhMYB108-GhRLF1-cytokinin (CTK) within the AZ to regulate cotton leaf abscission. GhRLF1 not only regulates leaf abscission but also reduces cotton yield. Consequently, transgenic lines exhibiting rapid leaf falling and requiring less defoliant, while maintaining unaffected cotton yield, were developed for mechanical harvesting. This was achieved using a defoliant-induced petiole-specific promoter proPER21, to drive GhRLF1 (proPER21::RLF1). This pioneering biotechnology offers a new strategy for the chemical defoliation of machine-harvested cotton, ensuring stable production and reducing leaf debris in harvested cotton, thereby enhancing environmental sustainability.