Cation of a provided molecules. The analyte concentrations, expressed as g-
Cation of a provided molecules. The analyte concentrations, expressed as g-1 dry weight (d.w.), have been calculated by comparison having a calibration curve obtained by utilizing a commercial normal of abietic acid (1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,two,three,four,4a,4b,five,six,10,10adecahydrophenanthrene-1-carboxylic acid (Sigma-Aldrich catalog N. 00010). The GC/MS approaches employed in the present study for the extraction and evaluation of plant metabolites have been adequately validated for their selectivity, precision, and efficiency. Selectivity was verified by observing that no interfering peak was apparent at the elution time of every single target analyte upon injecting 3 replicate blank samples. Precision was tested by measuring the inter- and intra-day variability in the chromatographic profiles of spiked samples, which ranged from 2 to 7 with regards to relative typical deviation. Ultimately, the intrinsic recovery in the extraction process was calculated as a mean of three replicate samples, in every single of which the plant tissue was spiked having a CK1 web identified aliquot of abietic acid common resolution and then extracted, cleaned, and derivatized before injection onto GC-MS. Irrespective of the tissue extracted, the measured imply recovery usually ranged from 80 to 90 . 3.three. RNA Isolation and cDNA Synthesis Total RNA was extracted from 250 mg of every single in the five tissues regarded as in accordance with Pavy et al. [40]. RNA concentration and integrity have been checked using a NanoDrop ND-1000 spectrophotometer (Labtech, East Sussex, UK). Only RNA samples having a 260/280 wavelength ratio in between 1.9 and 2.1, in addition to a 260/230 wavelength ratio higher than 2.0, had been made use of for cDNA synthesis. First-strand cDNA was synthesized from 3 of total RNA of every on the five tissues using a Xpert cDNA Synthesis Kit (GRiSP Research Answer, Porto, Portugal) based on the manufacturer’s instructions. three.four. DNA Extraction Genomic DNA was extracted from 100 mg of young and mature needles employing a NucleoSpinPlant II kit (Macherey-Nagel, D en, Germany) as outlined by the manufacturer’s guidelines. The integrity and concentration of DNA were determined by 0.8 (w/v) agarose gels stained with ethidium bromide (0.001 ) utilizing known concentrations of unrestricted lambda DNA as manage. three.five. Isolation of Partial and Full-Length cDNAs Coding for Diterpene Synthases In accordance with the methods reported in Alicandri et al. [20], RT-polymerase chain reaction (PCR) was used to amplify partial cDNA coding for DTPSs in P. nigra subsp. laricio by utilizing forward and reverse primers designed in conserved regions among DTPS sequences of Pinus species in the distinct Calcium Channel Inhibitor Species groups identified by phylogenetic analysis. The total list with the applied forward and reverse primers is reported in Table S1. Every PCR reaction was performed inside a total volume of 50 containing two of RT reaction obtained from a pool of total RNA from the 5 various tissues (see Section three.3), 0.four of each forward and reverse primer, and 25 of Xpert Taq Mastermix (2X) (GRiSPPlants 2021, 10,14 ofResearch Solutions, Porto, Portugal), which contains pure Xpert Taq DNA Polymerase, dNTPs, MgCl2 and optimized PCR buffer. All reactions were carried out in an Eppendorf Thermal Cycler (Master cycler Gradient) with all the following parameters: initial denaturation at 95 C for 5 min, 35 cycles of amplification, every at 95 C for 1 min, 582 C (based on the annealing temperature on the primers) for 1 min, 72 C for three min, and a final extension at 72 C for 5 min.