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)

Abstract

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.

Overexpression and RNAi-mediated Knockdown of Two 3β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis

“…Exogenous progesterone did also not enhance cardenolide formation in D. lanata wild type shoots [46]. Progesterone can be regarded as a reactive electrophile species (RES, [14]) which can be deactivated by P5βRs [6] and also by 3βHSDs, if we assume subsequent conjugation of the 4-pregnen-3-ol-20-one to substituents such as sugars or carboxylic acids, as previously seen [47] that might prevent oxidation back to progesterone. It is possible that the excess progesterone not used for cardenolide biosynthesis, may be funnelled into other metabolic processes.…”

“…It is possible that the excess progesterone not used for cardenolide biosynthesis, may be funnelled into other metabolic processes. The influence of exogenous progesterone has been discussed in some detail previously [14]. As P5βRs gene expression seemed to be slightly upregulated after progesterone treatment this effect may correspond to the detoxification reactions mentioned above, e.g., the conversion of progesterone by P5βRs.…”

“…Amplification of "megaprimers" and cloning PCR were conducted in a FlexCycler2 thermocycler (Analytik Jena) using PhusionR High-Fidelity DNA Polymerase (New England Biolabs). The protocol of RF-cloning followed the procedure previously described by [14]. E. coli SoluBL21 (DE3) cells (New England Biolabs), transformed with the pDEST17 vector, were grown on LB-medium containing 100 µg/mL ampicillin.…”

“…In addition, 3βHSD-like [12] as well as P5βR-like e.g. [13][14][15][16] genes are also known from noncardenolide plants and that the encoded enzymes may be used in other biosynthetic pathways [15]. These enzymes may thus be regarded as promiscuous enzymes with a potential of playing important roles in pathway evolution and the detoxification of xenobiotics and reactive carbonyl species [14,15].…”

Occurrence and conversion of progestogens and androgens are conserved in land plants

“…PO, first reported in plants in the 1960s (Bennett & Heftmann, 1965;Gawienowski & Gibbs, 1968), influences hypocotyl elongation and stress responses (Iino et al, 2007;Genisel et al, 2013;Shpakovski et al, 2017;Hao et al, 2019) and PO-binding proteins have been reported (Yang et al, 2005). Progestogens, especially PO, are a precursor for 5b-cardenolides (Klein et al, 2022). Despite advancements in characterizing plant progestogens and androgens (Janeczko, 2021), their evolutionary origin, biosynthesis, and physiological functions are largely unknown.…”