Abstract
Ethylene is an important plant hormone whose production relies on the action of key enzymes, one of which is 1-aminocyclopropane-1-carboxylate synthase (ACS). There are three classes of ACS, which are all partially regulated by degradation through the ubiquitin-proteasome system (UPS), which regulates ethylene production. Arabidopsis has a single class III ACS, ACS7, but although it is known to be degraded by the 26S proteasome, the UPS proteins involved are poorly characterised. In this work, we used mass spectrometry to identify novel components of the ubiquitin system that may contribute to the regulation of ethylene biosynthesis via ACS7. We found two HECT-type ligases, UPL1 and UPL2, which regulate ACS7 stability. In vitro experiments showed that UPL1 and UPL2 E3 ligases directly control ACS7 turnover. In addition, increased ethylene levels were observed in UPL1- and UPL2-knockout plants in response to NaCl and NaCl+MG132 treatment, respectively. Under the same conditions, we observed increased ACS7 transcript levels in upl1 compared to WT plants under control and stress conditions, further confirming that UPL1 and UPL2 regulate ACS7-dependent ethylene production in response to stress. We used molecular modelling to predict ACS7 ubiquitylation sites and cell-free degradation assays to verify that lysine residues at positions 174, 238 and 384 regulate ACS7 protein stability. Overall, this study provides new insights into the regulation of ACS7 protein stability, and hence ethylene production, in plant growth and development and the response to stress.