Characterization and localization of a wound-inducible type I serine-carboxypeptidase from leaves of tomato plants (Lycopersicon esculentum Mill.)

During the course of characterization of the wound-response related proteins from tomato (Lycopersicon esculentum Mill.) leaves, a serine carboxypeptidase (EC 3.4.16.1) was identified. An increase in peptidase activity in response to wounding, and the isolation of a protein with carboxypeptidase (CP) activity from tomato leaves had been reported previously, but the mRNA coding for the enzyme was not identified. We now report the isolation of a tomato leaf type I serine-CP cDNA whose corresponding mRNA is induced by wounding, systemin and methyl jasmonate. The protein sequence deduced from the cDNA exhibits homology to tomato CP, and barley and rice type I CPs. Southern blot results indicated that the CIP gene is probably a member of a small gene family. Tomato CP mRNA was detected within 3 h after wounding, or treatment with systemin or methyl jasmonate. Employing Western blot analysis, CP protein was shown to increase 12 h after the treatments. Using the tomato def1 mutant, we have demonstrated that a functional octadecanoid pathway is necessary for CP transcription in response to wounding. Carboxypeptidase protein was immunolocalized as protein aggregates within the central vacuoles of palisade mesophyll cells as well as in vascular parenchyma where it had previously been found. Double labeling using antibodies specific for CP and inhibitor II indicated that the two proteins are colocalized in the vacuolar aggregates. Tomato CP is a member of the "late wound-inducible genes" whose mRNAs increase 4-12 h following wounding, in contrast to several "early wound-inducible genes", whose mRNAs appear within 30 min. The data support a role for the enzyme in protein turnover that occurs systemically in leaf cells in response to wounding.