Quantification of Soluble Sugars in Leaves

Soluble sugars were extracted and quantified as before [61 (link)]. Ground leaf material (100 mg) was extracted in 1 mL of ddH₂O by boiling for 15 min at 95 °C. Samples were vortexed and centrifuged for 10 min at 15,000× g and 200 µL of supernatant applied to a Dowex^® (Acros Organics, Morris Plains, NJ, USA) anion and cation exchange column. Columns were subsequently rinsed 6 times with 200 µL of ddH₂O. The flow through was diluted 1:1 in 20 µM rhamnose H₂O as the internal standard. Samples were analysed by HPAEC-IPAD Dionex 5000 (Thermo Scientific, Waltham, MA, USA) with separation on a CarboPac^TM PA100 column (Thermo Scientific) and a mobile phase of 90 mM NaOH as described [50 (link)]. Concentrations were calculated using standards of 10 µM of each sugar run alongside.