Ubiquitome profiling reveals a regulatory pattern of UPL3 with UBP12 on metabolic-leaf senescence.

The HECT-type UPL3 ligase plays critical roles in plant development and stress protection, but understanding of its regulation remains limited. Here, the multi-omics analyses of ubiquitinated proteins in <i>upl3</i> mutants were performed. A landscape of UPL3-dependent ubiquitinated proteins is constructed: Preferential ubiquitination of proteins related to carbon fixation represented the largest set of proteins with increased ubiquitination in the <i>upl3</i> plant, including most of carbohydrate metabolic enzymes, BRM, and variant histone, whereas a small set of proteins with reduced ubiquitination caused by the <i>upl3</i> mutation were linked to cysteine/methionine synthesis, as well as hexokinase 1 (HXK1) and phosphoenolpyruvate carboxylase 2 (PPC2). Notably, ubiquitin hydrolase 12 (UBP12), BRM, HXK1, and PPC2 were identified as the UPL3-interacting partners in vivo and in vitro. Characterization of <i>brm</i>, <i>upl3</i>, <i>ppc2</i>, <i>gin2</i>, and <i>ubp12</i> mutant plants and proteomic and transcriptomic analysis suggested that UPL3 fine-tunes carbohydrate metabolism, mediating cellular senescence by interacting with UBP12, BRM, HXK1, and PPC2. Our results highlight a regulatory pattern of UPL3 with UBP12 as a hub of regulator on proteolysis-independent regulation and proteolysis-dependent degradation.