Subtilase-mediated maturation of EPF1 and EPF2 is crucial for stomatal patterning

Proper stomatal distribution optimizes the balance between acquiring atmospheric CO₂ for photosynthesis and minimizing water loss^1,2. In Arabidopsis, the EPIDERMAL PATTERNING FACTOR (EPF)-ERECTA family signalling pathway specifies this patterning^2-6. 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.