Non-structural carbohydrate analysis
We adapted methods from Mullin et al . (2021) for sugar and starch
analyses. Briefly, ground samples were extracted with 1.6 mL ultra-pure
water in a 2 mL glass tube. We placed tubes in a rack and enclosed the
rack in steam above a tray of boiling water for 60 min and then closed
the tube with its cap, and vortexed them for 30 sec. The resulting
extract was separated into two 0.5 mL aliquots for soluble sugars and
starch and stored at +4 °C.
For soluble sugars (glucose, fructose, sucrose), tubes containing a 0.5
mL aliquot were centrifuged at 18,213 rcf for 15 min and the resulting
supernatant was transferred into new 2 mL tubes containing 1.0 ml of
methanol and vortexed for 30 sec. Samples were incubated at room
temperature for 90 min and centrifuged at 18,213 rcf for 10 min.
Afterwards, 0.5 ml of extracts were transferred to 2 ml glass vials and
stored at −40 °C. Sugars were analyzed using an Ultra High-Performance
Liquid Chromatography (UHPLC, 1290 Infinity Agilent Tech., Santa Clara,
CA, USA) fitted with an InfinityLab Poroshell 120 HILIC-Z column (2.1 x
100 mm 2.7µm, Agilent Tech.) and an Evaporative Light Scattering
Detector (ELSD, 1290 ELSD II, Agilent Tech.). A gradient analysis was
performed using a binary solvent system of ultra-pure water with 0.02%
v/v ammonium hydroxide (channel A) and acetonitrile (HPLC-grade) with
0.02% v/v ammonium hydroxide (channel B) flowing at 0.2 ml
min-1. We calculated quadratic standard curves from
three dilutions prepared from analytical standards to quantify glucose
(Chemical Purity: 99%), fructose (CP: 99%), and sucrose (CP: 99.5%).
Soluble sugars were identified based on the retention time of the
analytical standards. Concentrations of soluble sugars were reported as
µg mg-1 of dry weight.
Starch extraction was conducted using the second aliquot (0.5 mL) of
sample extract. We used a series of enzymatic digestions to convert
starch into gluconate-6-phosphate as adapted from Lahr & Sala (2014)
and Cale et al. (2019a). Extractions began with the steam bath
procedure described above. Following the steam bath, samples were
vortexed for 30 sec, then a 0.5 ml aliquot that included suspended
solids was transferred to a new 2 mL tube containing 0.5 mL of α-amylase
solution (0.75 mg enzyme mL−1; Sigma-Aldrich) to
convert starch to maltose and similar polysaccharides. The tubes were
immediately vortexed for 30 sec, incubated in a water bath at 50° C for
16 hm and then centrifuged (18,213 rcf) for 20 min. The resulting 0.5 mL
of the supernatant was transferred into a new 2 mL tube containing with
0.5mL of amyloglucosidase solution 2.5 g enzyme (Sigma-Aldrich) in 50 mL
sodium acetate buffer (0.1 M, pH 4.5). The tubes were incubated in a 50
°C bath for 16 h to convert maltose and similar polysaccharides to
glucose and fructose and then centrifuged (18,213 rcf) for 15 min. We
transferred 0.02 mL of the final glucose extracts to 2 mL glass vials
with starch derived glucose, which were immediately quantified by
spectrophotometric analysis using the Synergy Microplate Reader H1
(BioTek, Winooski, VT, USA) at an absorbance of 340 nm. Using methods
reported in Mullin et al . (2021), we quantified the concentration
of starch using two calibration curves. The first curve estimated
glucose concentrations from the sample absorbance at 340 nm, and the
second estimated starch concentration from glucose concentration.