Nodulin cleavage by the cysteine protease CYP35 promotes soybean root nodule senescence

In legumes, symbiotic root nodules undergo senescence in response to developmental or environmental cues. This process determines the maintenance and nitrogen-fixing capacity of the root nodules, but the molecular mechanisms underlying its initiation are poorly understood. The cysteine protease CYP35 is a positive regulator of nodule senescence in soybean (Glycine max), but its substrates remain unknown. Here, we demonstrate that CYP35 promotes nodule senescence by cleaving a subset of Nodule-Enriched Nodulin proteins (NENs). Sequence and phylogenetic analyses indicate that CYP35 is a cathepsin L-like cysteine protease, with Cys149 as a key catalytic residue. CYP35 physically interacts with a distinct subfamily of 8 NENs, NEN1-8. Soybean quadruple and quintuple nen mutants obtained by multiplex gene editing develop nodules with accelerated senescence and reduced nitrogenase activity, whereas over-expression of NEN2 or NEN5 delays senescence and enhances nodule function. CYP35 proteolyzes NEN2, NEN5, NEN6, and NEN7 in vitro and cleaves NEN2 in vivo in a Cys149-dependent manner. Our findings establish a direct molecular link between cysteine protease-mediated Nodulin cleavage and the onset of nodule senescence in soybean, providing insights into the regulation of nodule lifespan and nitrogen fixation.