Analysis of non-structural carbohydrate concentrations
All tissues harvested for NSC and isotope analyses were dried in an oven at 65°C for 5 d. After drying, each sample was ground with a Retsch MM 300 ball mill (Retsch, Germany) until finely and homogeneously ground. NSCs are defined here as low-molecular-weight sugars and starch, and analysis followed the protocol by Schönbeck et al. (2018). About 10 mg of the sample powder was first vortexed with 2 ml of deionized water and then boiled in the steam for 30 min. For free-sugar analysis, a 200 μl aliquot of the extract was treated with invertase and isomerase (in 0.4 M Na-acetate buffer; Sigma-Aldrich, St. Louis, MO, USA) to break down sucrose to fructose and glucose. For the total NSCT(NSCT = soluble sugars + starch) analysis, a 500 µl aliquot of the extract (sugars and starch) was incubated with a fungal amyloglucosidase from Aspergillus niger (Sigma-Aldrich, St Louis, MO, USA) for 15 h at 49°C to digest starch into glucose. Both soluble sugars and NSCT concentrations were determined at 340 nm in a 96-well microplate photometer (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA) after enzymatic conversion of glucose molecules derived from sugars and starch to gluconate-6-phosphate (via isomerase, hexokinase, and glucose-6-P dehydrogenase; all supplied by Sigma-Aldrich). NSC concentrations are expressed as a percentage of dry matter, and the concentration of starch was calculated as NSCT minus free sugars.