Degreening of Citrus unshiu fruits via ethylene-induced soluble chlorophyllase

The mechanism of ethylene action on degreening in satsuma mandarin (Citrus unshiu Mere. cv. Nichinan No. 1) fruits was investigated using inhibitors o f protein synthesis and soluble chlorophyllase (chlorophyll- chlorophyllide hydrolase, Chlase; E.C. 3.1.1.14). This enzyme which catalyzes the dephyty lation of chlorophyll (Chl) to chlorophyllide (Chlide), was partially purif ied. Chlase catalyzes the first step in the Chi breakdown process. Ethylene trea tments promoted the degreening and increased soluble Chlase activity of C. unshiu fruit peels held in the dark. Cycloheximide (CH), an inhibitor of cy toplasmic protein synthesis, inhibited the effects of ethylene, whereas chl oramphenicol (CP), an inhibitor of plastid protein synthesis, also weakly i nhibited the Chlase activity. These results suggest that ethylene appears t o enhance the degreening of the peels through de novo synthesis of Chlase. Moreover, de novo cytoplasmic protein synthesis and chloroplast- dependent enzyme synthesis may regulate Chlase activity in the peel of ethylene- trea ted C. unshiu fruits. Soluble Chlase was precipitated with acetone from C. unshiu fruit peels and purified by the following method: after ammonium sulfate fractionation, ge l-filtration through Sephadex G-25 and Phenyl Sepharose CL-4B (hydrophobic chromatography) were used. The soluble Chlase was purified ca. 203.5-fold w ith a yield of 45.5%.