0.22 μm pore size nylon membrane

Cytokinins (trans-zeatin, t-Z, zeatin riboside, ZR, and isopentenyl adenine, iP), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and gibberellins (GA₁, GA₃, and GA₄) were analyzed according to Albacete et al. (2008) (link) with some modifications. Briefly, xylem sap samples were filtered through 13 mm diameter Millex filters with 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA). Ten microliter of filtrated extract were injected in a U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive spectrometer (ThermoFisher Scientific, Waltham, MA, USA) using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the plant hormones, calibration curves were constructed for each analyzed component (1, 10, 50, and 100 μg l^-1).

The Targas-1 equipment (PP Systems, Amesbury, MA, USA) was used to determine photosynthetic traits, including the leaf internal concentration of CO₂ (Ci: μmol mol⁻¹), photosynthesis rate (A: μmol CO₂ m⁻² s⁻¹), and stomatal conductance (gs: mmol H₂O m⁻² s⁻¹), following the instructions provided in the user manual. Measurements were conducted on the second youngest leaf from four plants per treatment, one day before harvest. To extract photosynthetic pigments, 0.5 g of fresh material was mixed with 5 mL of methanol for 24 h. The resulting samples were then filtered using 13 mm diameter Millex filters with a 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA). The absorbance of the filtered liquid was measured at wavelengths of 663 nm and 645 nm, corresponding to chlorophyll A and B, respectively, using a Synergy H1 Hybrid Multi-Mode microplate spectrophotometer (BioTek Inc., Winooski, VT, USA). The concentrations of pigments were calculated using the provided formula by Lichtenthaler [106 ].