Computational protocol: Identification and localization of bioactive naphthoquinones in the roots and rhizosphere of Paterson’s curse (Echium plantagineum), a noxious invader

Similar protocols

Protocol publication

[…] Solid phase root zone extraction (SPRE) microprobes (5cm) were prepared as per using silicone polydimethylsiloxane (PDMS) Silastic tubing (Fisher Scientific, USA), supported internally with fine stainless steel wire (22 gauge), as a probe for entrapment of nonpolar soil rhizosphere PSPs such as NQs. To fully evaluate whether microprobes could be used to entrap and collect NQs to intensify concentrations of these compounds in a rhizosphere setting, eight sets of three microprobes each were placed in contact with live roots collected from mature greenhouse-grown plants. Roots were sectioned into 5cm long pieces and placed in contact with microprobes for 1 or 12h in each of eight sterile Petri dishes containing moistened filter paper. After treatment, unexposed microprobes (those not subjected to root exposure and therefore serving as a negative control) and root-exposed microprobes were photographed. All unexposed controls and those microprobes exposed to roots for 12h were then extracted in HPLC grade 100% ethanol (VWR Chemicals, Australia) for 10min, followed by evaporation of extracts under a stream of N2 gas for approximately 20min. The resulting dried extract was weighed and resuspended in ethanol, filtered through a 22 μm Millex syringe filter and subjected to UHPLC/Q-ToF MS analysis as described below.Greenhouse-grown plants were propagated for 16 weeks in a soil mix containing 6:4 peat potting mix:sand (Scotts Co., Melbourne, Australia.). Seedlings were pre-germinated by imbibing in sterile water for 1 week using field-collected seed as described above in NSW, Australia; after 7 d, seedlings were transplanted into 1.5-l pots and were maintained in the glasshouse at 25/18 °C day/night temperatures at ~55% relative humidity. Plants were watered every other day by subirrigation and fertilized once per fortnight using a commercial liquid fertilizer (N:P:K=23:3.95:14, Aquasol Soluble Fertilizer, Australia). At 14 weeks of age, as plants began to flower, plants were sampled for rhizosphere-produced NQs using 5cm microprobes, as described above. Eight probes were placed equidistantly into each of six plant pots and were fully inserted into the soil surrounding the living plant, ~8cm from the centre of the plant rosette. The probes were placed equidistantly around the plant at a distance of ~5cm from the taproot, and were not in contact with the foliage of the plant.In addition, soil media was collected separately from the rhizosphere (soil not adhering to plant roots but located around living plant root system) of each pot, thoroughly filtered to remove any small residual root pieces using a fine wire mesh screen (<1mm mesh holes), air-dried and extracted in 100% ethanol, filtered using a 22 μm Millex filter and subjected to analysis using an ion trap mass spectrometer for NQ detection. [...] Abundance of deoxyshikonin, shikonin, acetylshikonin and dimethylacrylshikonin was analysed in 21 samples, representing 21 populations. Analysis of variance was performed on log transformed data in IBM SPSS statistics software (IBM Corp., NY, USA). Homogeneity of variances was assessed using Levene’s test prior to further analysis. Tukey HSD was used as a post hoc test to evaluate differences among metabolite levels averaged over populations. […]

Pipeline specifications

Software tools AquaSol, SPSS
Applications Miscellaneous, Protein physicochemical analysis
Chemicals Naphthoquinones