Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

Synthesis and cholinesterase inhibition of cativic acid derivatives.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with memory impairment and cognitive deficit. Most of the drugs currently available for the treatment of AD are acetylcholinesterase (AChE) inhibitors. In a preliminary study, significant AChE inhibition was observed for the ethanolic extract of Grindelia ventanensis (IC50=0.79 mg/mL). This result prompted us to isolate the active constituent, a normal labdane diterpenoid identified as 17-hydroxycativic acid (1), through a bioassay guided fractionation. Taking into account that 1 showed moderate inhibition of AChE (IC50=21.1 µM), selectivity over butyrylcholinesterase (BChE) (IC50=171.1 µM) and that it was easily obtained from the plant extract in a very good yield (0.15% w/w), we decided to prepare semisynthetic derivatives of this natural diterpenoid through simple structural modifications. A set of twenty new cativic acid derivatives (3-6) was prepared from 1 through transformations on the carboxylic group at C-15, introducing a C2-C6 linker and a tertiary amine group. They were tested for their inhibitory activity against AChE and BChE and some structure-activity relationships were outlined. The most active derivative was compound 3c, with an IC50 value of 3.2 µM for AChE. Enzyme kinetic studies and docking modeling revealed that this inhibitor targeted both the catalytic active site and the peripheral anionic site of this enzyme. Furthermore, 3c showed significant inhibition of AChE activity in SH-SY5Y human neuroblastoma cells, and was non-cytotoxic.

Allocation of biomass and photoassimilates in juvenile plants of six Patagonian species in response to five water supply regimes.

BACKGROUND AND AIMS: The growth-differentiation balance hypothesis (GDBH) states that there is a physiological trade-off between growth and secondary metabolism and predicts a parabolic effect of resource availability (such as water or nutrients) on secondary metabolite production. To test this hypothesis, the response of six Patagonian Monte species (Jarava speciosa, Grindelia chiloensis, Prosopis alpataco, Bougainvillea spinosa, Chuquiraga erinacea and Larrea divaricata) were investigated in terms of total biomass and resource allocation patterns in response to a water gradient. METHODS: One-month-old seedlings were subjected to five water supply regimes (expressed as percentage dry soil weight: 13 %, 11 %, 9 %, 7 % or 5 % - field water capacity being 15 %). After 150 d, plants were harvested, oven-dried and partitioned into root, stem and leaf. Allometric analysis was used to correct for size differences in dry matter partitioning. Determinations of total phenolics (TP), condensed tannins (CT), nitrogen (N) and total non-structural carbohydrates (TNC) concentrations were done on each fraction. Based on concentrations and biomass data, contents of TP and CT were estimated for whole plants, and graphical vector analysis was applied to interpret drought effect. KEY RESULTS: Four species (J. speciosa, G. chiloensis, P. alpataco and B. spinosa) showed a decrease in total biomass in the 5 % water supply regime. Differences in dry matter partitioning among treatments were mainly due to size variation. Concentrations of TP, CT, N and TNC varied little and the effect of drought on contents of TP and CT was not adequately predicted by the GDBH, except for G. chiloensis. CONCLUSIONS: Water stress affected growth-related processes (i.e. reduced total biomass) rather than defence-related secondary metabolism or allocation to different organs in juvenile plants. Therefore, the results suggest that application of the GDBH to plants experiencing drought-stress should be done with caution, at least for Patagonian Monte species.