The ethylene-induced chlorophyll catabolism of radish (Raphanus sativus L.) cotyledons: Production of colorless fluorescent chlorophyll catabolite (FCC) in vitro

Abstract

Ethylene enhanced the degreening and increased the chlorophyll (Chl)-oxidative enzyme activity in intact radish (Raphanus sativus L.)(Rs) cotyledons kept in the dark. The catabolism of Chl a-porphyrin in plants was investigated by using crude proteins from ethylene-induced degreening radish cotyledons. The enzyme catalyzes the degradation of Chl a in the presence of H2 O2 and 2,4-dichlorophenol (DCP), or p-coumaric acid. Analysis by HPLC and HPTLC revealed the appearance of C-132-hydroxy (HO)Chl a. The reaction of C-132-HO Chl a formation was inhibited completely by ascorbate (2mM) or KCN (2mM), but it was not under an anoxygenic condition. These results suggest that C-132-HO Chl a forming enzyme is a peroxygenase and/or peroxygenase-like peroxidase, the peroxidase catalyzed Oxygen transfer from H2 O2. Moreover, the 3 dimensional (3-D) fluorescence spectral measurements showed that a colorless fluorescent Chl catabolite (FCC: Ex 350 nm/Em 455 nm) was produced with a concomitant decrease in Chl a level. The production of C-132-HO Chl a and colorless Rs-FCC was inhibited by heating the extracts. It appears that Chl a-porphyrin is catabolized by the proteins prepared from Raphanus sativus cotyledons via the following reaction: Chl a → C-132HO Chl a → → colorless Rs-FCC (Chl-porphyrin oxygenolytic cleavage catabolite).