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.