Restoring cardenolide accumulation in RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata and studying the effects of precursor feeding on cardenolide free dark-cultured D. lanata shoots

Progesterone 5β-reductases (P5βR) catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata (Dl). RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines provided experimental evidence for the participation of P5βRs in 5β-cardenolide formation [1]. These lines show reduced cardenolide (ca. 25 - 30%) levels as compared with wildtype shoots of the same shoot culture clone (set to equal 100%). Progesterone levels in RNAi-mediated knockdown lines were decreased compared to WT shoots and cardenolides levels could not be restored by exogenously added progesterone. We here tried to restore cardenolide content of the knockdown lines by feeding 5β-pregnane-3,20-dione, a cardenolide precursor downstream of progesterone. We observed a recovery of cardenolide content in Dl P5βR1-RNAi lines whereas the level in the controls was not further increased.

Cardenolide content in Dl P5βR1-RNAi lines reached around 2/3 (0.8 μmol×g−1 DW) of the cardenolide level of WT shoots. Still high GSH levels in Dl P5βR2-RNAi lines seem to continuously disturb cardenolide formation. Cardenolide levels only increased from 0.3 μmol*g ^− 1 DW to 0.5 μmol*g⁻¹ DW.

Besides using a system of RNAi-mediated knockdown lines, we established cardenolid-free D. lanata shoot cultures by dark cultivation to study exogenously feed precursor effects on cardenolide formation [2]. Feeding 5β-pregnane-3,20-dione to D. lanata shoots kept in dark, cardenolide content remained unchanged low, but GSH and hydrogen peroxide levels decreased, indicating a reduced stress level.

Publikationsverlauf

Artikel online veröffentlicht:
13. Dezember 2021

• References

• 1 Klein J, Horn E, Ernst E, Leykauf T, Leupold T, Dorfner M, Wolf L, Ignatova A, Kreis W, Munkert J. RNAi-mediated gene knockdown of progesterone 5β-reductases in Digitalis lanata reduces 5β-cardenolide content. Plant Cell Reports, accepted.
• 2 Eisenbeiß M, Kreis W, Reinhard E. Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures. Plant Physiology and Biochemistry, Volume 37, Issue 1, 1999, Pages 13-23.