2.2. Quantification of Indole-3-Acetic Acid (IAA)

Quantification of indole-3-acetic acid (IAA) quantification was carried out following the method proposed by Rawlinson et al. [29] with some modifications. Arabidopsis roots were cut with a sharp blade below the hypocotyl, and were immediately frozen in liquid nitrogen, ground to a fine powder and were aliquoted. Further, weighed amounts of the powder (100 mg) per treatment and replicate were poured into 2 mL microcentrifuge tubes for extraction. To the samples, 20 µL of a 20 mg/mL solution of 3-indolepropionic acid (IPA) was added as internal standard for quantification and normalization purposes. Successively, 200 µL of NaOH (1% w/v), 147 µL of methanol (MeOH) and 34 µL of pyridine were added and the samples were vortexed for 40 s. To the extracted samples 20 µL of methyl chloroformate were added and samples were again vortexed for 30 s (and this step was repeated twice). To this extract, 400 µL of chloroform was added, samples were shaken for 10 s and 400 µL of a NaHCO₃ solution (50 mM stock) was added. Samples were shaken again for 20 s and were immediately centrifuged (14,000 rpm) for 1 min. The organic lower phase was collected and dispensed into a new 2 mL centrifuge tube and the aqueous residues were eliminated using anhydrous Na₂SO₄. An aliquot (100 µL) of this organic phase was used for gas chromatography mass spectrometry (GC-MS) analysis. A parallel experiment was carried out using pure IAA (Sigma Aldrich, 20149, Milano, Italy, Cat. No. I3750-25G-A) as an external standard for retention time (RT) assignment and for the generation of a standard curve.

GC-MS analysis was carried out using a Thermo Fisher (Thermo Fisher Scientific, 20090 Rodano (MI), Italy) gas chromatograph apparatus (Trace 1310) equipped with a single quadrupole mass spectrometer (ISQ LT). The capillary column (MEGA-5MS 30 m × 0.25 mm × 0.25 µm) was connected to a 10 m long pre-column (MEGA S.r.l., 20025 Legnano (MI), Italy), and the carrier gas was helium with a flow rate of 1 mL/min. Injector and transfer line were set at 250 °C and 270 °C, respectively. Samples (3 µL) were injected with a 35 psi pressure pulse, which was held for 1 min. The following GC temperature program was used: Isocratic for 1 min. at 40 °C, from 40 °C to 320 °C with a rate of 20 °C min⁻¹, then held isocratic for 2 min at 320 °C. The ion source was set to 200 °C and the solvent delay was 4.5 min. Mass spectra were recorded in electronic ionization (EI) mode at 70 eV, scanning at 50–400 m/z range to select appropriate EI mass fragments for each analyte. Then the MS was run in selected ion monitoring (SIM) using one quantifier (m/z) and two qualifiers (m/z) ions. In particular, for IAA-methyl ester the ions 189, 103, 77 were selected for quantification. IAA identification and quantification were performed by comparing the RT with the IAA external standard and the mass spectra in the National Institute Standard and Technology (NIST 2011) spectral library. The relative IAA quantification was carried out normalizing the IAA peak intensity with the intensity of the internal standard.