SUMO-specific proteases SPF1 and SPF2 negatively regulate seed oil synthesis by mediating WRI1 deSUMOylation.

SUMO PROTEASE RELATED TO FERTILITY 1 (SPF1) and SPF2 are responsible for deSUMOylation of SUMO-conjugated protein substrates and for maintaining protein SUMOylation homeostasis. To date, the role of SUMO proteases in fatty acid biosynthesis and lipid accumulation remains unclear. Here, we demonstrate that the Arabidopsis thaliana mutantsspf1-1, spf2-1, and spf1-1 spf2-1 exhibit increased seed size and elevated seed oil content (SOC). We further show that SPF1 and SPF2 interact with WRINKLED1 (WRI1), a master regulator of the transcriptional control of plant oil synthesis. Genetic analyses indicate that the spf1-1 wri1-3 and spf2-1 wri1-3 double mutants, as well as the spf1-1 spf2-1 wri1-3 triple mutant, phenocopy wri1-3 and display severe seed shriveling, indicating that SPF1 and SPF2 act upstream of WRI1. WRI1 was identified as a SUMO1 substrate with two conserved SUMOylation sites, lysine 257 (K257) and K266, in cruciferous plants, with K257 acting as the dominant site required for seed oil synthesis. SUMOylation enhances WRI1 stability, whereas SPF1- and SPF2-mediated deSUMOylation promotes WRI1 degradation. In spf1-1, spf2-1, and spf1-1 spf2-1 mutants, the abundance of SUMOylated WRI1 increases during seed development and correlates with elevated seed oil accumulation. Together, these results indicate that SPF1 and SPF2 negatively regulate oil synthesis by deSUMOylating WRI1, establishing a dynamic SUMOylation and deSUMOylation switch centered on the SPF1/SPF2-WRI1 module that fine-tunes seed development and oil synthesis.