NGS Transcriptomes and Enzyme Inhibitors Unravel Complexity of Picrosides Biosynthesis in Picrorhiza kurroa Royle ex. Benth

Abstract

Picrorhiza kurroa is an important medicinal herb valued for iridoid glycosides, Picroside-I (P-I) and Picroside-II (P-II), which have several pharmacological activities. Genetic interventions for developing a picroside production platform would require knowledge on biosynthetic pathway and key control points, which does not exist as of today. The current study reports that geranyl pyrophosphate (GPP) moiety is mainly contributed by the non-mevalonate (MEP) route, which is further modified to P-I and P-II through phenylpropanoid and iridoid pathways, in total consisting of 41 and 35 enzymatic steps, respectively. The role of the MEP pathway was ascertained through enzyme inhibitors fosmidomycin and mevinolin along with importance of other integrating pathways using glyphosate, aminooxy acetic acid (AOA) and actinomycin D, which overall resulted in 17%-92% inhibition of P-I accumulation. Retrieval of gene sequences for enzymatic steps from NGS transcriptomes and their expression analysis vis-à-vis picrosides content in different tissues/organs showed elevated transcripts for twenty genes, which were further shortlisted to seven key genes, ISPD, DXPS, ISPE, PMK, 2HFD, EPSPS and SK, on the basis of expression analysis between high versus low picrosides content strains of P. kurroa so as to eliminate tissue type/ developmental variations in picrosides contents. The higher expression of the majority of the MEP pathway genes (ISPD, DXPS and ISPE), coupled with higher inhibition of DXPR enzyme by fosmidomycin, suggested that the MEP route contributed to the biosynthesis of P-I in P. kurroa. The outcome of the study is expected to be useful in designing a suitable genetic intervention strategy towards enhanced production of picrosides. Possible key genes contributing to picroside biosynthesis have been identified with potential implications in molecular breeding and metabolic engineering of P. kurroa.

Molecular Regulation of Catalpol and Acteoside Accumulation in Radial Striation and non-Radial Striation of Rehmannia glutinosa Tuberous Root

“…Based on precursor feeding experiments, researchers established the pathway of catalpol biosynthesis and inferred that catalpol and other iridoid glycosides were generated from geraniol, which was produced through a combined biosynthetic route involving non-mevalonate (MEP) and mevalonate (MVA) pathways [19,20]. Shitiz et al reported that the complete biosynthetic pathway of catalpol has been deciphered for all possible intermediates with their corresponding enzymes in Picrorhiza kurroa (Figure 6A) [21]. The putative genes encoding enzymes in the upstream of the iridoid pathway, including geraniol synthase (GES), geraniol 10-hydroxylase (G10H), cytochrome P450 reductase (CPR), and 10-hydroxygeraniol oxidoreductase (10HGO), have been identified in R. glutinosa [16].…”

Metabolic and transcriptional analyses in response to potent inhibitors establish MEP pathway as major route for camptothecin biosynthesis in Nothapodytes nimmoniana (Graham) Mabb

“…In Salvia miltiorrhiza , it has been reported that MVA pathway flux plays main role in cell growth, whereas the intermediates of MEP pathway are the major source of tanshinone biosynthesis [20]. Similarly, in Picrorhiza kurroa the role of the MEP pathway in picroside biosynthesis was determined by using enzyme inhibitors mevinolin and fosmidomycin along with aminooxy acetic acid, glyphosate and actinomycin D [31]. In N. nimmoniana camptothecin production involves a complex network of pathways including MEP, MVA, shikimate and seco-iridoid pathways.…”

“…Moreover, MEP has been reported as a major route for production of seco-iridoid moiety secologanin in C. roseus [29] and taxol in Taxus baccata [30]. Similarly, in Picrorhiza kurroa , by using different enzyme inhibitors (fosmidomycin, mevinolin, glyphosate and aminooxy acetic acid) and transcriptional inhibitor actinomycin D, confirmed that non-mevalonate pathway is the main source of picroside biosynthesis [31]. In Arabidopsis , transient reduction of sterol levels were observed in presence of HMGR inhibitor (lovastatin), indicating that the plastidial MEP pathway compensates for the lack of cytosolic IPP required for synthesis of cytosolic sterols [32].…”

Exogenous feeding of immediate precursors reveals synergistic effect on picroside-I biosynthesis in shoot cultures of Picrorhiza kurroa Royle ex Benth

“…The biosynthesis of P-I proceeds via non-mevalonate (MEP), mevalonate (MVA), shikimate/phenylpropanoid and iridoid pathways 5 . During the past years, numerous efforts have been carried out to shortlist candidate genes involved in P-I production through gene expression analyses performed under differential conditions of picrosides accumulation in P. kurroa 6 7 8 9 10 11 12 . Despite interesting cues obtained from these reports, no information is yet available on what mechanism controls the flux of P-I biosynthesis in P. kurroa .…”