Immunocytolocalisation of ACC Oxidase in Tomato Fruits

Abstract

ACC oxidase has long been considered as an insoluble membrane-bound enzyme. It is now clear that it is in fact soluble [1], but its subcellular localization has not been elucidated yet. However, we had demonstrated earlier that plant cells can generate ethylene from either apoplastic or intracellular ACC, indicating that two potential sites exist for ethylene formation [2].The discovery that the pTOM13 cDNA was encoding EFE in tomato fruits [3] opened new possibilities for the study of ACC oxidase. Two types of polyclonal antibodies were raised against: (i) a synthetic peptide (VEKEAZEESTQUY) considered as a suitable epitope by secondary structure predictions and (ii) a recombinant ACC oxidase polypeptide overproduced in E. coli from pTOM13. Antibodies were purifed by affinity column chromatography and eventually saturated with excess of antigenes. Pericarp tissue of both normal and transgenic tomatoes transformed with antisense pTOM13 cDNA (kindly provided by A Hamilton and D. Grierson, Nottingham, UK) were processed for immunocytolocalization as described in [4].