Regulation of NADPH-cytochrome P450 reductase expressed during Douglas-fir germination and seedling development

NADH-cytochrome P450 is a key enzyme that transfers electrons from NADPH to the cytochrome P450 family of enzymes. To begin to determine the regulation of CPR gene expression and enzyme activity in Douglas-fir a full-length cDNA was isolated from a seedling lambda ZAP cDNA library and the ORF was used to develop a synthetic CPR-peptide-based antiserum. Northern blot analysis indicated CPR expression was regulated both developmentally prior to seed maturation and during germination, and differentially in the cotyledons, radicle and megagametophyte of seed and seedling tissues. The CPR-peptide antiserum detected a single CPR in seed and seedling microsomes analyzed by western blot of two-dimensional SDS-polyacrylamide gels. In microsomal extracts from whole seeds and seedlings, the amount of CPR protein remained constant while NADPH:cytochrome c reductase activity increased during stratification, germination and early seedling development. In contrast to cotyledons and megagametophyte, the level of CPR protein detected in radicles was higher than expected when compared to the amount of CPR transcript.