Abstract
Key messageStudying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-cardenolide formation.Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5βRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5β-cardenolide formation, we here established several RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5βR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5βR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5βR2-RNAi lines but not in Dl P5βR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5βR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).
Highlights
The successful introduction of foxglove extracts to treat congestive heart failure in the eighteenth century (Withering 1941) made plants from the genus Digitalis (Plantaginaceae) worth investigating (Luckner and Wichtl 2000).1 3 Vol.:(0123456789)Plant Cell Reports (2021) 40:1631–1646In the twentieth century cardiac glycosides, the bioactive principles of Digitalis plants replaced the use of extracts
To eliminate the possibility that effects attributed to gene knockdown may have been provoked by stress, we investigated the influence of glutathione (GSH), buthionine sulfoximine (BSO), methyl vinyl ketone (MVK), and progesterone on cardenolide formation and progesterone 5β-reductase gene expression in wild-type and knockdown shoots
MVK was accepted much better by rDlP5βR1 than by rDlP5βR2 indicating that DlP5βR1 and DlP5βR2 may play different roles within the plant
Summary
The successful introduction of foxglove extracts to treat congestive heart failure in the eighteenth century (Withering 1941) made plants from the genus Digitalis (Plantaginaceae) worth investigating (Luckner and Wichtl 2000).1 3 Vol.:(0123456789)Plant Cell Reports (2021) 40:1631–1646In the twentieth century cardiac glycosides, the bioactive principles of Digitalis plants replaced the use of extracts. Other medications have superseded Digitalis in the treatment of heart failure, cardenolides came back into focus as they showed anti-viral and anti-tumoral activity (Prassas and Diamandis 2008; Bertol et al 2011; Schneider et al 2017). Today cardenolides such as digoxin, digitoxin, and lanatoside C are still isolated from Digitalis lanata Ehrh. P5βR-like enzymes (termed iridoid synthases; ISY) are involved in iridoid biosynthesis (Geu-Flores et al 2012), and Munkert et al (2015a, b) reported that iridoid synthase activity is common among the plant progesterone 5β-reductase family. PRISEs seem to be involved in reactions in central plant metabolism and appeared early in plant evolution, presumably with lateral gene transfer originating from α-proteobacteria (Tarrío et al 2011)
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