Phosphorylation of tomato 1-aminocyclopropane-1-carboxylic acid synthase, LE-ACS2, at the C-terminal region.

1-aminocyclopropane-1-carboxylic acid synthase is a key enzyme in the ethylene biosynthesis pathway. Recent studies raise the possibility that 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is regulated not only transcriptionally but also post-translationally. To elucidate post-translational ACS regulation, we analyzed the modification of LE-ACS2 protein, a wound-inducible isozyme in the ACS family, in tomato fruit (Lycopersicon esculentum L.) using an anti-LE-ACS2 antibody. We detected phosphorylated LE-ACS2 at 55-kDa using immunoprecipitation from an extract of wounded fruit fed with [32P]inorganic phosphate. Analysis of LE-ACS2 phosphoamino acids indicated that serine residue(s) were phosphorylated. In vitro phosphorylation analyses using site-directed mutagenesis of recombinant LE-ACS2 as a substrate demonstrate that serine 460 located at the C-terminal region of ACS is phosphorylated. During tomato ripening stages, expression of both LE-ACS2 and LE-ACS4 mRNA increased. LE-ACS4, however, was not phosphorylated in vitro. These results suggest that ACS isozymes have different post-translational regulatory mechanisms, such as phosphorylation.