Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures

Shoot cultures of the cardenolide-producing species Digitalis lanata Ehrh. accumulated up to 0.6 mu mol cardenolides per g dry mass when cultivated un der continuous white light. After transfer to permanent dark, the cardenoli de content of cultured shoots gradually decreased and reached non-detectabl e levels after 12 weeks. After transfer back to light conditions, cardenoli des started to accumulate and reached the levels of light-grown controls af ter 4 weeks. Radiolabelled pregnenolone and progesterone were incorporated into cardenolides in both green light-grown and white dark-grown shoots. It was thus established that cardenolides are synthesised de novo in chloropl ast-free tissues without apparent cardenolide accumulation, indicating that these compounds are efficiently turned over in the dark and that tissue di fferentiation, but not intact chloroplasts, is essential for cardenolide fo rmation. The time course of two late anabolic enzymes of cardenolide metabo lism, acetyl-CoA:digitoxin 15'-O-acetyltransferase (DAT, EC 2.3.1.-) and UD P-glucose:digitoxin 16'-glucosyltransferase (DGT, EC 2.4.1.-) was establish ed during transfer of shoots from light to dark and vice versa. Only DAT wa s affected and was not measurable any more under dark conditions. The DGT m ay not be down-regulated because of its important, maybe even vital, role a s an enzyme providing the vacuolar storage forms of cardenolides. Two catab olic cardenolide-specific enzymes, lanatoside 15'-O-acetylesterase (LAE, EC 3.1.1.6.) and cardenolide 16'-O-glucohydrolase I (CGH I, EC 3.2.1.21), wer e also investigated and it was demonstrated that CGH I is inactive in dark- grown shoots. These observations indicate that CGH I is not involved in car denolide degradation in situ, but may instead play a role in cardenolide re metabolisation and activation after wounding or in developmental programs. (C) Elsevier, Paris.