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Digestive proteolysis executed by the proteasome plays an important role in plant development. Yet, the role of limited proteolysis in this process is still obscured due to the absence of studies. Previously, we showed that limited proteolysis by the caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) modulates development in plants through the cleavage of unknown substrates. Here we used a modified version of the positional proteomics method COmbined FRActional DIagonal Chromatography (COFRADIC) to survey the proteolytic landscape of wild-type and separase mutant RADIALLY SWOLLEN 4 (rsw4) root tip cells, as an attempt to identify targets of separase. We have discovered that proteins involved in the establishment of pH homeostasis and sensing, and lipid signalling in wild-type cells, suggesting novel potential roles for separase. We also observed significant accumulation of the protease PRX34 in rsw4 which negatively impacts growth. Furthermore, we observed an increased acetylation of N-termini of rsw4 proteins which usually comprise degrons identified by the ubiquitin-proteasome system, suggesting that separase intersects with additional proteolytic networks. Our results hint to potential pathways by which separase could regulate development suggesting also novel proteolytic functions.