Lc net 2 adc

The three different pools of fructans (Fruct1, Fruct2, and Fruct3) were studied by high performance size-exclusion chromatography (HPSEC) as described in Dos-Santos et al. [23 (link)]. The average molecular weight (MW) was measured in a Jasco equipment (LC-Net II ADC, Kyoto, Japan) with a refractive index detector (Jasco RI-1530) and injection valve (Rheodyne, loop 20 μL, Cotati, CA, USA). A TSKgel G3000PWXL column (300 × 7.8 mm i.d., Tosoh Bioscience GmbH, Griesheim, Germany) was used after calibration with 70, 40, 6 kDa, maltotriose, sucrose and glucose (Fluka, Buchs, Switzerland). The elution was performed at a flow rate of 1 mL/min. The regression equation to calculate the average MW of fructan pools was: $$t_{elut}=-1.614logMW+16.681,$$
with a R² value of 0.9918. The average DP was calculated by dividing the calculated MW by 162.

The presence and quantification of tricin, ρ-coumaric acid, ferulic acid, cinnamic acid, and salicylic acid by high-performance liquid chromatography (HPLC) analyses were compared with the standards attained by the method presented by Anh et al. [33 ]. In brief, the HPLC system consisted of a pump (PU-4180 RHPLC, Jasco, Tokyo, Japan), a controller (LC-Net II/ADC, Jasco, Japan), and a detector (UV-4075 UV/VIS, Jasco, Tokyo, Japan). A column (130 Å, 5 µm, 2.1 × 100 mm) (XBridge BEH Shield RP18, Waters Cooperation, Milford, MA, USA) was used as a stationary phase. Solvent A (0.1% formic acid in water) and solvent B (acetonitrile) were applied as mobile phases, which were fixed in the same gradient program reported by Anh et al. [33 ]. Each operation was continued for 35 min at room temperature. Every sample was identified by a corresponding peak scanned at 350 nm for tricin and 280 nm for ρ-coumaric acid, ferulic acid, cinnamic acid, and salicylic acid. The peak area was used to quantify these compounds.

Solutions of 10 mg/mL of each sample were analyzed by HPLC [21 (link)]. Briefly, the analyses were carried out using a Jasco-LC-Net II ADC liquid chromatograph system equipped with a diode array detector (DAD). Phenols were separated using a Mediterranea Sea C18 reverse-phase analytical column (25 cm length × 4.6 mm i.d., 5 μm particle size; Teknokroma, Barcelona, Spain). The gradient profile was formed using solvent A (water with 1% formic acid) and solvent B (acetonitrile with 1% formic acid): from 0% B to 20% B for the first 20 min, to 21% B over the next 8 min, maintained at 21% B for 2 min, and then to 30% B over the next 10 min, and to 100% over the next 5 min, and finally maintained at 100% B for 5 min. The flow rate was 1 mL/min and the column temperature was 30 °C. Spectra from all peaks were recorded in the 200–600 nm range and the chromatograms were acquired at 360 nm. The quantification of individual phenols was performed using an eight-point regression curve in the range of 0–250 μg on the basis of standards.

Exudates were collected from the total root system of each plant following the methodology described in Delgado et al. (2014) (link). Briefly, the roots were washed with tap water, incubated in CaSO₄ (0.2 mM) and shaken for 2 h. Subsequently, the solution was filtered, to avoid the presence of microorganisms, with a sterile syringe containing a filter of 0.22 μm. The liquid samples containing the exudates were frozen at −20°C and then lyophilized using a freeze-dryer (Model FD8508, Bondiro, Ilshin Lab, Co. Ltd., Korea). Finally, lyophilized samples were re-suspended in water for high-performance liquid chromatography (HPLC) and quantified using HPLC equipment (JASCO, LC-Net II/ADC, Tokyo, Japan) following the protocol described by Delgado et al. (2013) (link). Citrate, malate, oxalate, and succinate were used as standards. These determinations were carried out at the Institute of Agroindustries of the Universidad de La Frontera, Temuco. The values were expressed as a rate of carboxylates exuded per gram of fresh weight (FW) per hour (μmol g^–1 FW h^–1). The exudates from six seedlings per species grown in the different substrates were analyzed.