Abstract
1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is the rate-limiting enzyme of the ethylene biosynthesis pathway. ACS is regulated both transcriptionally and post-translationally. We previously reported that LeACS2 is phosphorylated in vivo, and suggested the possibility that phosphorylation regulates protein stability rather than enzymatic activity. Here, we demonstrate that phosphorylation/dephosphorylation of LeACS2 regulates its stability. Pulse-chase experiments coupled with treatment with protein kinase/phosphatase inhibitors clearly demonstrated that LeACS2 is stabilized by phosphorylation and immediately degraded after dephosphorylation. The amount of LeACS2 affected by the protein kinase/phosphatase inhibitors significantly influenced cellular ACS activity, ACC content, and ethylene production levels in tomato fruit tissue, suggesting that post-translational regulation by phosphorylation plays an important role in the control of ethylene production as well as in transcriptional regulation. LeACS2 was immediately phosphorylated after translation by CDPK and MAP kinase at different sites in response to wound signaling. CDPK inhibitor, calmidazolium, and MAPKK inhibitor, U0126, were decreased the amounts of phosphorylated form and total ACS protein. Furthermore, the remaining ACS proteins were phosphorylated at both sites; suggesting that type 1 ACS required both phosphorylation sites to be stabilized.
