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

Abstract

In the current study, we asked how the supply of immediate biosynthetic precursors i.e. cinnamic acid (CA) and catalpol (CAT) influences the synthesis of picroside-I (P-I) in shoot cultures of P. kurroa. Our results revealed that only CA and CA+CAT stimulated P-I production with 1.6-fold and 4.2-fold, respectively at 2.5 mg/100 mL concentration treatment. Interestingly, feeding CA+CAT not only directed flux towards p-Coumaric acid (p-CA) production but also appeared to trigger the metabolic flux through both shikimate/phenylpropanoid and iridoid pathways by utilizing more of CA and CAT for P-I biosynthesis. However, a deficiency in the supply of either the iridoid or the phenylpropanoid precursor limits flux through the respective pathways as reflected by feedback inhibition effect on PAL and decreased transcripts expressions of rate limiting enzymes (DAHPS, CM, PAL, GS and G10H). It also appears that addition of CA alone directed flux towards both p-CA and P-I production. Based on precursor feeding and metabolic fluxes, a current hypothesis is that precursors from both the iridoid and shikimate/phenylpropanoid pathways are a flux limitation for P-I production in shoot cultures of P. kurroa plants. This work thus sets a stage for future endeavour to elevate production of P-I in cultured plant cells.

Transcriptome Analysis of Bael (Aegle marmelos (L.) Corr.) a Member of Family Rutaceae

“…RNA-Seq targeting expressed coding sequences has been used successfully in many medicinal and non-model plant species that do not have a reference genome (e.g., Prosopis cineraria L. [39], Andrographis paniculata Burm.f. [40], Phyllanthus emblica L. [41], Picrorhiza kurroa [42], and Azadirachta indica Royle ex Benth. [43]).…”

Modular Design of Picroside-II Biosynthesis Deciphered through NGS Transcriptomes and Metabolic Intermediates Analysis in Naturally Variant Chemotypes of a Medicinal Herb, Picrorhiza kurroa

“…Conversely, 150 μM aqueous solution of VA (Sigma-Aldrich, USA) was mixed with 70 μM CAT (Sigma-Aldrich, USA) and used for the feeding of in vitro grown P. kurroa shoots. These concentrations were selected based on their effects on P-I production in P. kurroa when applied alone and in combination under tissue culture conditions ( Kumar et al, 2016b ). Following this, the solutions of different precursors were added into the culture tubes containing optimized MS medium [containing 3 mg/L indole-3-butyric acid (IBA) and 1 mg/L kinetin (KN)] after filter sterilization through 0.22 μm syringe filter (Millipore) ( Sharma et al, 2015 ).…”

“…It has been hypothesized that the catalpol (CAT) backbone derived from iridoid pathway undergoes esterification reactions with trans -cinnamic acid (CA) and vanillic acid (VA) to produce P-I and P-II, respectively ( Kumar et al, 2013 ). The metabolic route of P-I biosynthesis has been ascertained through precursor feeding and enzyme inhibitor studies along with the assortment of key candidate genes through expression analysis using qRT-PCR vis-à-vis P-I content ( Pandit et al, 2013b ; Kumar et al, 2015b , 2016b , c ; Shitiz et al, 2015 ). However, the complexity of P-II biosynthesis is still unresolved as both protocatechuate and ferulic acid (FA); the metabolites of shikimate/phenylpropanoid pathway, are plausible to produce VA, the immediate precursor of P-II ( Figure 1 ) ( Kawoosa et al, 2010 ; Gahlan et al, 2012 ; Kumar et al, 2013 ).…”

Production of Medicinal Compounds from Endangered and Commercially Important Medicinal Plants through Cell and Tissue Culture Technology for Herbal Industry

“…The concept is based on the idea that any compound which is an intermediate, or is in the beginning of a secondary metabolite biosynthetic route, proves to be a good candidate for increasing the final yield of secondary metabolite. Varun et al [49] has carried out exogenous feeding of immediate biosynthetic precursor, i.e., cinnamic acid and catalpol in the shoot cultures of Picrorhiza kurroa hence stimulated 4.2 fold production of picroside-1. The production of monoterpene alkaloids was increased in cell suspension cultures of Catharanthus roseus fed with precursor mevalonic acid, secologanin [50].…”