Overexpression of 3β-hydroxysteroid dehydrogenases in Digitalis lanata shoot cultures

Leykauf, Tim; Klein, Jan; Lödel, M; Kreis, Wolfgang; Munkert, Jennifer

doi:10.1055/s-0041-1736957

Cited by 1 publication

(3 citation statements)

References 5 publications

(6 reference statements)

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“…All species examined had more than one Ec3βHSD -like gene, with the exception of E. cheiranthoides . The three orthologs from E. crepidifolium (44), two from D. lanata (30–32), and two from A. thaliana , AtSDR5 (AT2G47140) and AtSDR3 (AT2G47130) (10), have been shown to accept cardenolide intermediates in vitro , but in planta evidence for involvement in cardenolide biosynthesis only exists for Dl3βHSD1 (30) (Figures 6A, S15). Of the species included in the KSI gene tree, only E. cheiranthoides contained more than one copy (Figures 6B, S16).…”

Section: Resultsmentioning

confidence: 99%

“…In this study, we identified and biochemically characterized four cardenolide biosynthetic enzymes from E. cheiranthoides . Of these, Ec3βHSD and EcP5βR2 belong to enzyme families that have been speculated, or shown, to be involved in cardenolide biosynthesis in other species (13, 30, 31, 45, 47). However, the mutant lines generated in this study provide critical in vivo evidence for their role in cardenolide synthesis, and the lack of a cardenolide phenotype for EcP5βR1 knockouts highlights the potential disparity between in vitro activity and in vivo function.…”

Section: Discussionmentioning

confidence: 99%

“…Enzymes of this type are thought to be required for cardenolide biosynthesis (30)(31)(32). as yellow rectangles.…”

Section: Mutant Screensmentioning

confidence: 99%

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Four enzymes control natural variation in the steroid core ofErysimumcardenolides

Younkin,

Alani,

Züst

et al. 2024

Preprint

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Plants commonly produce families of structurally related metabolites with similar defensive functions. This apparent redundancy raises the question of underlying molecular mechanisms and adaptive benefits of such chemical variation. Cardenolides, a class defensive compounds found in the wallflower genusErysimum(L., Brassicaceae) and scattered across other plant families, show substantial structural variation, with glycosylation and hydroxylation being common modifications of a steroid core, which itself may vary in terms of stereochemistry and saturation. Through a combination of chemical mutagenesis and analysis of gene coexpression networks, we identified four enzymes involved in cardenolide biosynthesis inErysimumthat work together to determine stereochemistry at carbon 5 of the steroid core: Ec3βHSD, a 3β-hydroxysteroid dehydrogenase, Ec3KSI, a ketosteroid isomerase, EcP5βR2, a progesterone 5β-reductase, and EcDET2, a steroid 5α-reductase. We biochemically characterized the activity of these enzymes in vitro and generated CRISPR/Cas9 knockout lines to confirm activity in vivo. Cardenolide biosynthesis was not eliminated in any of the knockouts. Instead, mutant plants accumulated cardenolides with altered saturation and stereochemistry of the steroid core. Furthermore, we found variation in carbon 5 configuration among the cardenolides of 44 species ofErysimum, where the occurrence of some 5β-cardenolides is associated with the expression and sequence of P5βR2. This may have allowedErysimumspecies to fine-tune their defensive profiles to target specific herbivore populations over the course of evolution.

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