MdACO expression during abscission: the use of 33P labeled primers in transcript quantitation

The last step of ethylene biosynthesis in apple is catalyzed by ACO (1-aminocyclopropane 1-carboxylic acid oxidase) encoded by MdACO1 and MdACO2. Both genes were expressed during early fruit development although at different level. Later on, the ACO transcript accumulation in persisting fruitlets decreased whereas in fruitlets undergoing abscission MdACO2 transcripts remained constant and those of MdACO1dramatically increased. The expression of MdACO was assessed by a particular type of semi-quantitative PCR involving 33P labeled primers and the following electrophoresis in polyacrylamide gel. The use of either the reverse primer or the anchored oligo dT13 produced clear and elegant results. Nevertheless, the initial quantity of labeled primer appeared to be a crucial factor for obtaining a wide range titration curve.