IMMUNOCYTOLOCALIZATION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE IN TOMATO AND APPLE FRUIT

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethyl ene, has been studied in ripening fruits of tomato (Lycopersicum escul entum Mill.). Two types of antibody have been raised against (i) a syn thetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overpro duced in Escerichia coli expressing the pRC13 cDNA. Immunoblot analysi s showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidas e. Superimposition of Calcofluor white with immunofluorescence labelli ng, analysed by optical microscopy, indicated that ACC oxidase is loca ted at the cell wall in the pericarp of breaker tomato and climacteric apple(Malus x domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Tr ansgenic tomato fruits in which ACC-oxidase gene expression was inhibi ted by an antisense gene exhibited a considerable reduction of labelli ng. Immunocytological controls made with pre-immune serum or with anti bodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protei n predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.