Study on mechanism of posttranslational regulation of ACC synthase by phosphorylation that involved in tomato fruit ripening

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ACC synthase (ACS) is a rate-limiting enzyme of ethylene biosynthesis pathway and each ACS isozyme expresses in response to various each stimulus. Recent studies suggested that ACS was regulated not only transcriptionally but also post-translationally. We found that LeACS2, a wound-inducible ACS in tomato (Lycopersicon esculentum Mill.), is immediately phosphorylated after translation at Ser-460 in the C-terminal region by calcium-dependent protein kinase (CDPK), and then acts in the phosphorylated form in the cell. Moreover, the results of treatments of kinase and phosphatase inhibitors showed that the half-life of phosphorylated LeACS2 was longer than that of non-phosphorylated LeACS2. These results suggest that phosphorylation/dephosphorylation regulates the turnover of LeACS2 protein in the cell and that dephosphorylation of LeACS2 causes degradation. Analyses of ethylene-overproducer (eto) mutants also supported our speculation. Thus, we attempted to identify the protein phosphatase involved in LeACS2 turnover. Biotin-tagged phospho-peptide (Biotinyl-KNNLRL(pS)FSKRMYD-CHO) based on the sequence in the neighborhood of Ser-460 of LeACS2 was synthesized. This peptide was incubated with the extract of wounded tomato fruit tissue. Proteins bound to the peptide were captured by streptavidin-bound Dynabeads and eluted by SDS. The eluate was subjected to SDS-PAGE and blotting, and detected with HRP conjugated streptavidin. As the results, 32 kDa protein was detected. The amount of the 32 kDa protein was dependent on the concentration of the phospho-peptide that was added to reaction mixture. Based on the molecular mass, we speculate the 32 kDa protein is a catalytic subunit of Ser/Thr protein phosphatase that is involved in dephosphorylation of LeACS2.