Multiple Forms of NADPH-Cytochrome P450 Oxidoreductases in the Madagascar Periwinkle Catharanthus roseus

NADPH-cytochrome P450 oxidoreductase (CPR, EC 1.6.2.4) is the redox partner of classical P450-monooxygenases, which have crucial roles in the metabolism of terpenes, alkaloids, flavonoids, phytoalexins, etc. It becomes evident that, contrary to animals and yeast, various CPR isoforms occur in some plants, although their specific physiological functions are largely unknown. C. roseus-CPK has been reported as encoded by a single gene and early papers concerning the C. roseus-CPR protein also reported a single CPR polypeptide. The observation of diverse CPRs during purification or by immunoblot were attributed to proteolytic degradation. We obtained CPR immunotype of C. roseus roots using two heterologous antisera directed against the CPR from Sorghum bicolor and Helianthus tuberosus, respectively. Both antisera developed the same immunogenic profile with two cross-reactive polypeptides. Further evaluation of anti-H. tuberosus CPR serum excluded non-specific binding of antiserum with C. roseus microsomal proteins. The two immuno-reactive polypeptides are probably not the result of proteolytic degradation, since increasing protease inhibitor concentration during the extraction and manipulation of the samples did not affect the occurrence of these two CPR forms. Roots from plants growing in the field showed identical CPR immunotypes with those seen in vitro, indicating that this immunoprofile actually belongs to C. roseus roots. The lectin concanavalin A was able to inhibit the CPR activity from C. roseus hairy roots; therefore, the immuno-reactive polypeptides probably result from post-translational glycosylation of the original polypeptide. Not only the roots, but also the flowers, leaves and the stem showed more than a single CPR form. The different tissues of the plant showed different immuno-reactive bands, which were reproducible even though they came from tissues of plants growing in the field. This opens the possibility of the occurrence of diverse tissue-specific CPRs.