AtRALF1 precursor processing in the Golgi complex involves two subtilisin-like serine proteases

AtSBT6.1 is the primary physiological convertase for AtRALF1, while AtSBT5.3 contributes under ectopic AtRALF1 overexpression. The 5-kDa rapid alkalinization factor (RALF) peptide is widespread in the plant kingdom. In Arabidopsis, the RALF family comprises 37 RALF-like peptides. AtRALF1 is a 49-amino-acid peptide derived from a 120-amino-acid precursor, but the protease responsible for its maturation has remained unresolved. We used in silico expression profiling to select subtilases (AtSBTs) potentially involved in AtRALF1 precursor processing. T-DNA insertion AtSBT mutants, and F3 homozygous lines derived from crosses between AtSBT mutants, and an AtRALF1-overexpressing line were analyzed for primary root length (PRL) and lateral root primordium (LRP) density. PRL and LRP density were higher in atsbt6.1 than in wild-type plants, whereas atsbt5.3 was indistinguishable from the wild-type. Ectopic expression of AtRALF1 driven by the 35S promoter in atsbt6.1 and atsbt5.3 backgrounds indicated that both AtSBT6.1 and AtSBT5.3 contribute to AtRALF1 precursor processing under overexpression conditions. In contrast, the phenotypes of single mutants indicate that AtSBT6.1 plays the predominant role in AtRALF1 precursor processing when AtRALF1 is expressed from its endogenous promoter. Transient expression assays further showed that AtRALF1 precursor processing occurs in the Golgi complex. These findings identify AtSBT6.1 as the main subtilase for AtRALF1 maturation in planta and reveal context-dependent participation of AtSBT5.3 under ectopic AtRALF1 expression.