Nucleodur c18 analytical column

The hormone content was measured as previously described [49 (link)]. Leaf samples (40 mg) were freeze-dried, homogenized, transferred to a 2-mL centrifuge tube, and extracted in an ultrasonic bath for 1 h with 1 mL of a methanol:water (1:1 v/v) mixture, acidified with 0.1% formic acid. After centrifuging the samples for 10 min at 15,000 rpm and 4 °C, the supernatant was used to quantify ABA, IAA, and SA, adopting the external standard technique, with calibration curves obtained using ABA (Sigma Aldrich, St Louis, MO, USA; purity 98.5%), IAA (Sigma Aldrich; purity ≥ 99%), and SA (Sigma Aldrich, purity ≥ 99%) original analytical standards. The HPLC-DAD equipment was an Agilent 1220 Infinity LC system model G4290B (Agilent^®, Waldbronn, Germany), which included a gradient pump, autosampler, and a 30 °C column oven. A 170 Diode Array Detector (Gilson, Middleton, WI, USA) was used with a Nucleodur C18 analytical column (250 × 4.6 mm i.d., 5 μm, Macherey Nagel) set at 265 nm for both ABA and IAA and at 280 nm for SA. The mobile phases were water acidified with 0.1% formic acid (A) and acetonitrile (B), at a flow rate of 0.600 mL min⁻¹ in gradient mode, 0–6 min: 30% of B, 6–16 min: from 30% to 100% B, 16–21 min: 100% B. Twenty μL per sample were injected, running three biological replicates for each condition; data are expressed as µg g⁻¹ of dry weight.

Hormone content was quantified as reported in Pagliarani et al. (2022) (link) and Nerva et al. (2022) (link). Following freeze drying, 40 mg of tissue was homogenized and extracted in an ultrasonic bath for 1 h with 1 ml of a mixture of methanol:water (1:1, v/v), acidified with 0.1% formic acid. After centrifugation (15,000 rpm, 10 min, 4°C), the supernatant was used to quantify abscisic acid (ABA), indole-3-acetic acid (IAA), and SA, adopting the external standard technique, with calibration curves made with original analytical standards (all from Sigma Aldrich, purity ≥98.5% for ABA and ≥99% for IAA and SA). The HPLC apparatus (Agilent 1220 Infinity LC system model G4290B, Agilent, Waldbronn, Germany) was equipped with a gradient pump, an autosampler, and a column oven set at 30°C. A 170 Diode Array Detector (Gilson, Middleton, WI, USA) set at 265 nm was employed, using a Nucleodur C18 analytical column (5 μm length: 250 mm, ID: 4.6 mm, Macherey-Nagel GmbH & Co. KG, Düren, Germany). The mobile phases were water acidified with 0.1% formic acid (A) and acetonitrile (B), at a flow rate of 0.600 ml min⁻¹ in gradient mode, 0–6 min: from 10% to 30% of B, 6–16 min: from 30% to 100% B, 16–21 min: 100% B. Twenty microliters per sample was injected, testing all biological replicates (n = 3).

Leaf veins (40 mg) were freeze dried and homogenised, then transferred in a 2 mL centrifuge tube and extracted with 1 mL of methanol: water (1:1 v/v) acidified with 0.1% of formic acid in an ultrasonic bath for 1 h. Samples were centrifuged at 15,000 rpm and 4 °C for 10 min, and the supernatant was analysed by HPLC-DAD technique. Original standard of ABA (purity ≥ 98.5%; Sigma-Aldrich) was used for metabolite identification by comparing retention times and UV spectra. Metabolite quantification was made by external calibration method. An Agilent 1220 Infinity LC system model G4290B (Agilent®, Waldbronn, Germany), equipped with gradient pump, autosampler and column oven set at 30 °C, and a 170 Diode Array Detector (Gilson, Middleton, The USA) set at 265 nm were employed. A Nucleodur C18 analytical column (250 × 4.6 mm i.d., 5 μm, Macherey Nagel) was used. The mobile phases consisted of water acidified with formic acid 0.1% (A) and acetonitrile (B), at a flow rate of 0.600 mL min⁻¹ in gradient mode, 0–6 min: 30% of B, 6–16 min: from 30 to 100% B, 16–21 min: 100% B. Twenty μL were injected for each sample and three biological replicates were run for each analysis.