RNAi-mediated gene knockdown of progesterone 5β-reductases in Digitalis lanata reduces 5β-cardenolide content

Abstract

Key message Studying 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. Abstract 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).

Plastidial Expression of 3β-Hydroxysteroid Dehydrogenase and Progesterone 5β-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco

“…The substrate for 3β-HSD is pregnenolone which is converted into progesterone due to enzymatic reaction. However, there are several substrates for the enzymes P5βR1 and P5βR2, such as 1,4-enones, such as 2-cyclohexen-1-one, methyl vinyl ketone or citral are also accepted [ 32 ] but progesterone exhibits more specificity as a substrate [ 3 ]. Secondary metabolites (cardenolides and progesterone) biosynthesis is also triggered due to abiotic (salt, wound, heat, cold) [ 3 ] or biotic stress factors (pathogen or insect herbivory) [ 33 ].…”

Knockout of Arabidopsis thaliana VEP1, Encoding a PRISE (Progesterone 5β-Reductase/Iridoid Synthase-Like Enzyme), Leads to Metabolic Changes in Response to Exogenous Methyl Vinyl Ketone (MVK)

“…Glutathione reductase (GR1) is an enzyme influencing the redox state of a cell by releasing reduced glutathione (GSH) from oxidized glutathione (GSSG) [34]. Redox states can therefore be evaluated by measuring GR1 expression [14].…”

“…PRISEs came into focus in specialized plant metabolism because they act as progesterone 5β-reductases in 5β-cardenolide biosynthesis [11][12][13][14] and iridoid synthases in iridoid biosynthesis [7,15]. RNAi-mediated gene knockdown of PRISEs led to reduced cardenolide contents in Digitalis lanata [14].…”

“…PRISEs came into focus in specialized plant metabolism because they act as progesterone 5β-reductases in 5β-cardenolide biosynthesis [11][12][13][14] and iridoid synthases in iridoid biosynthesis [7,15]. RNAi-mediated gene knockdown of PRISEs led to reduced cardenolide contents in Digitalis lanata [14]. The direct participation of PRISEs in iridoid biosynthesis was demonstrated in virus-induced gene silencing (VIGS) experiments using Catharanthus roseus [15].…”

“…MVK is a powerful inducer of defense-related gene expression [21] and PRISEs may therefore modify stress responses through metabolizing MVK and related compounds. Consequently, a more general physiological role for PRISEs has been proposed in stress responses and detoxification processes [9,14].…”