Auxin Quantification in Leaf Tissue

For auxin measurements, 100 mg fresh weight of leaf tissue (leaves 8–12) per sample was collected and shock-frozen in liquid nitrogen under white light (time points during light exposure) or green safety light (time points during night). Analysis was carried out using high-performance liquid chromatography–electrospray tandem mass spectrometry as described in [67 (link)] using 10 mg tissue per biological replicate. Samples were extracted with 1 mL of 50 mM phosphate buffer (pH 7.0) containing 0.1% sodium diethyldithiocarbamate. [¹³C₆]IAA, [¹³C₆]oxIAA, [¹³C₆]IAA-Glc, [¹³C₆]oxIAA-Glc, [¹³C₆]IAA-Asp and [¹³C₆]IAA-Glu (5 pmol of each) were added as internal standards. A 200 µL portion of extract was acidified with 1M HCl to pH 2.7 and purified by in-tip micro solid-phase extraction (in-tip µSPE). After evaporation under reduced pressure, samples were analyzed using HPLC system 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) equipped with Kinetex C18 column (50 mm × 2.1 mm, 1.7 µm; Phenomenex) and linked to 6495 Triple Quad detector (Agilent Technologies, Santa Clara, CA, USA). Auxin levels were quantified using stable-isotope-labeled internal standards as a reference.