Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM-N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and th e synthesis of catabolites as did the addition to BM-N of cycloheximide or a chelator, 2,2'-bipyridyl. In contrast, chloramphenicol or the protease in hibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II , cytochrome f and protochlorophyllide oxido-reductase. During growth in BM -N, protease activity increased and proteins immunologically detectable wit h an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM-N and BM+N cells were used for differential and subtractive s creening to isolate cDNAs representing genes with degreening-enhanced expre ssion (dee) in C. protothecoides. Several different dees were identified wi th different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM-N cultures. Analysis of th e dee sequences showed that they encode different proteins including a nove l amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca2+- binding protein, MO25, ubiquinone-cytochrome c-reductase and several new pr oteins.