Proper stomatal distribution optimizes the balance between acquiring atmospheric CO2 for photosynthesis and minimizing water loss1,2. In Arabidopsis, the EPIDERMAL PATTERNING FACTOR (EPF)–ERECTA family signalling pathway specifies this patterning2–6. Similar to most signalling peptides, EPF1 and EPF2 require proteolytic processing to convert inactive precursor proteins into functional mature peptides7; however, in contrast to the well-characterized signalling cascade downstream of the ERECTA family2, little is known about the mechanisms of proteolytic release of the mature EPF1/2 from precursors. Here we identify a group of subtilisin-like serine proteinases8, designated as EPF-PROCESSING PROTEINASES (EPPs), which play a crucial role in stomatal patterning by processing EPF1 and EPF2 precursors. Loss-of-function mutations in EPPs lead to a dramatic increase and clustering of stomata, resembling the phenotypes observed in epf1/2 and er-105 erl1/2 mutants. Notably, these defects can be mitigated through the exogenous application of mature EPF1/2 peptides. Moreover, mutations of these EPPs inhibit the cleavage of EPF1/2 precursors and attenuate their associated overexpression phenotypes. Furthermore, biochemical assays demonstrate that EPPs cleave EPF1/2 both in vitro and in vivo. Taken together, our findings elucidate the molecular mechanisms underlying EPP-mediated processing of EPF1/2 precursors. This proteolytic release of active peptides is identified as a critical, previously missing link required for establishing proper stomatal patterning in plants.
