Small or specialized natural products (SNAPs) produced by plants vary greatly in structure and function, leading to selective advantages during evolution. With a limited number of genes available, a high promiscuity of the enzymes involved allows the generation of a broad range of SNAPs in complex metabolic networks. Comparative metabolic studies may help to understand why—or why not—certain SNAPs are produced in plants. Here, we used the wound-induced, vein patterning regulating VEP1 (AtStR1, At4g24220) and its paralogue gene on locus At5g58750 (AtStR2) from Arabidopsis to study this issue. The enzymes encoded by VEP1-like genes were clustered under the term PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) as it was previously demonstrated that they are involved in cardenolide and/or iridoid biosynthesis in other plants. In order to further understand the general role of PRISEs and to detect additional more “accidental” roles we herein characterized A. thaliana steroid reductase 1 (AtStR1) and compared it to A. thaliana steroid reductase 2 (AtStR2). We used A. thaliana Col-0 wildtype plants as well as VEP1 knockout mutants and VEP1 knockout mutants overexpressing either AtStR1 or AtStR2 to investigate the effects on vein patterning and on the stress response after treatment with methyl vinyl ketone (MVK). Our results added evidence to the assumption that AtStR1 and AtStR2, as well as PRISEs in general, play specific roles in stress and defense situations and may be responsible for sudden metabolic shifts.

Summary: Progestogens and androgens have been found in many plants, but little is known about their biosynthesis and the evolution of steroidogenesis in these organisms. Here, we show that the occurrence and biosynthesis of progestogens and androgens are conserved across the viridiplantae lineage. An UHPLC‐ESI‐MS/MS method allowed high‐throughput analysis of the occurrence and chemical conversion of progestogens and androgens in 41 species across the green plant lineage. Dehydroepiandrosterone, testosterone, and 5α‐dihydrotestosterone are plants' most abundant mammalian‐like steroids. Progestogens are converted into 17α‐hydroxyprogesterone and 5α‐pregnane‐3,20‐dione. Androgens are converted into testosterone and 5α‐dihydrotestosterone. 17,20‐Lyases, essential for converting progestogens to androgens, seem to be most effective in monocot species. Our data suggest that the occurrence of progestogens and androgens is highly conserved in plants, and their biosynthesis might favor a route using the Δ⁴ pathway.

5β-Cardenolides are pharmaceutically important metabolites of the specialized metabolism of Digitalis lanata. They were used over decades to treat cardiac insufficiency and supraventricular tachycardia. Since the 1960s, plant scientists have known that progesterone is an essential precursor of cardenolide formation. Therefore, plant progesterone biosynthesis was mainly analyzed in species of the cardenolide-containing genus Digitalis during the following decades. Today, Digitalis enzymes catalyzing the main steps of progesterone biosynthesis are known. Most of them were found in a broad range of organisms. This review will summarize the findings of 60 years of research on plant progesterone metabolism with a particular focus on the recent results in Digitalis lanata and other plants.

Progestogens and androgens have been found in many plants, but little is known about their physiological function. We used a previously established UPLC-ESI-MS/MS method to analyze progestogen and androgen profiles in fungal infections. Here we show that dehydroepiandrosterone (DHEA), a C₁₉ steroid, specifically accumulates in shoots of Arabidopsis thaliana (L.) HEYNH. infected with Alternaria brassicicola (SCHWEIN.) WILTSHIRE. Elevated DHEA levels in plants seem not to be product of fungal sterol/steroid precursor activity, but an intrinsic plant response to the infection. DHEA was applied exogenously to analyze the effects of the androgen on development and gene expression in A. thaliana. Our findings reveal that DHEA treatment downregulates membrane-associated, salicylic acid and abscisic acid-regulated, as well as stress-responsive genes. Notably, DHEA does not inhibit the isoprenoid or post-lanosterol pathway of the ergosterol biosynthesis. Moreover, A. brassicicola was also treated with DHEA to analyze the growth, sterol pattern and membrane-integrity. Our data suggest that DHEA enhances the permeability of plant and fungal biomembranes. We propose that DHEA accumulation is a plant defense response which reduces fungal growth in plant tissues.

Enzymes capable of processing a variety of compounds enable plants to adapt to diverse environmental conditions. PRISEs (progesterone-5β-reductase/iridoid synthase-like enzymes), examples of such substrate-promiscuous enzymes, are involved in iridoid and cardenolide pathways and demonstrate notable substrate promiscuity by reducing the activated C=C double bonds of plant-borne and exogenous 1,4-enones. In this study, we identified PRISE genes in Plantago media (PmdP5βR1) and Plantago lanceolata (PlP5βR1), and the corresponding enzymes were determined to share a sequence identity of 95%. Despite the high sequence identity, recombinant expressed PmdP5βR1 was 70 times more efficient than PlP5βR1 for converting progesterone. In order to investigate the underlying reasons for this significant discrepancy, we focused on specific residues located near the substrate-binding pocket and adjacent to the conserved phenylalanine “clamp”. This clamp describes two phenylalanines influencing substrate preferences by facilitating the binding of smaller substrates, such as 2-cyclohexen-1-one, while hindering larger ones, such as progesterone. Using structural analysis based on templates PDB ID: 5MLH and 6GSD from PRISE of Plantago major, along with in silico docking, we identified positions 156 and 346 as hot spots. In PlP5βR1 amino acid residues, A156 and F346 seem to be responsible for the diminished ability to reduce progesterone. Moreover, the double mutant PlP5βR_F156L_A346L, which contains the corresponding amino acids from PmdP5βR1, showed a 15-fold increase in progesterone 5β-reduction. Notably, this modification did not significantly alter the enzyme’s ability to convert other substrates, such as 8-oxogeranial, 2-cyclohexen-1-one, and methyl vinyl ketone. Hence, a rational enzyme design by reducing the number of hotspots selectively, specifically improved the substrate preference of PlP5βR1 for progesterone.

SUMMARY: Progestogens and androgens are steroids found in a wide range of plants, but little is known about their physiological functions. In this study, we sowed seeds of angiosperms on progestogen‐ and androgen‐containing medium and analysed their morphological effects. We further investigated the effects of progesterone and testosterone on brassinosteroid profiles and gene expression in A. thaliana. Additionally, we examined the effects of progesterone and testosterone on A. thaliana plants overexpressing the steroid 5α‐reductase DET2. We found that progestogens and androgens have strong negative effects on root length, especially in Brassicaceae species. In addition, these steroids led to uncoordinated cell growth and increased lateral root formation. We failed to detect an effect on endogenous brassinosteroid levels and gene expression of brassinosteroid‐regulated genes. The overexpression of DET2 led to increased root growth, but the effects of progesterone and testosterone were not reduced. We conclude that progestogens and androgens act in a brassinosteroid‐independent manner. This suggests that progestogens and androgens could represent a potential new class of plant steroid signalling molecules.