ETHYLENE INDUCES DE-NOVO SYNTHESIS OF CHLOROPHYLLASE, A CHLOROPHYLL DEGRADING ENZYME, IN CITRUS-FRUIT PEEL

Chlorophyllase (Chlase; EC 3.1.1.14) was extracted from plastid fracti ons of ethylene-treated orange fruit peel and purified 400-fold to hom ogeneity by gel filtration, hydrophobic chromatography, and preparativ e SDS/PAGE of nonheated protein. SDS/PAGE of nonheated purified enzyme indicated that Chlase activity is associated with a single protein ba nd migrating at an apparent molecular mass of 25 kDa whereas the heate d purified enzyme had a molecular mass of 35 kDa. The N-terminal seque nce of the purified protein was determined. The purified enzyme was us ed as an immunogen for raising antibodies in rabbits. The antiserum wa s highly specific and on Western blots recognized both the heated and the nonheated form of Chlase. The antibodies also recognized the solub ilized enzyme, as shown by an immunoprecipitation assay and by antigen -antibody capture assays in microtiter plates. Treatment with ethylene , which enhances degreening, increased Chlase activity 12-fold. Immuno blot analyses of crude extracts from ethylene-treated fruit detected a strong signal of the Chlase protein, while only a trace level of the enzyme protein could be detected in air. Gibberellin A3 and N6-benzyla denine partly counteracted the ethylene-induced increase in Chlase act ivity as well as the immunodetected upsurge of the Chlase protein. Eth ylene appears to enhance the degreening of citrus fruit through de nov o synthesis of the Chlase protein, which in turn is inhibited by the s enescence-delaying regulators, gibberellin A3 and N6-benzyladenine. Th e Chlase enzyme protein may, therefore, serve as a model system for st udying the hormonal molecular regulation of fruit ripening and senesce nce.