Quantification of Stem Sugars and Starch

Roughly 25 mg of air‐dried stem sample was weighed in a 2 ml tube and extracted twice at 85°C with a total of 2 ml of 80% ethanol. The supernatant was collected in a new 2 ml tube and was re‐extracted with 50 mg activated charcoal (Sigma) to eliminate pigments that interfere with sugar analysis. A 1 ml aliquot of the pigment free extract was incubated overnight in a heating block maintained at 50°C, and the resulting pellet was dissolved in 120 μl of water. A 10 μl aliquot was used for estimation of sucrose and glucose using assay kits (Sigma). Starch from the pellet was digested using 1 U of α‐amylase (from Aspergillus oryzae, Sigma) and amyloglucosidase (from Aspergillus niger, Sigma). After starch removal, the pellet was dried overnight at 95 °C and used for estimating structural sugars. Roughly, 5 mg of sample was weighed in a 2 ml tube and digested with 50 μl of 75% v/v H₂SO₄ for 60 min. The reaction was diluted by adding 1.4 ml of water, and tubes were sealed using lid‐locks and autoclaved for 60 min in a liquid cycle. After cooling, the sample was neutralized with CaCO₃ and sugar composition was estimated using high performance liquid chromatography (HPLC, LaChrom Elite® system, Hitachi High Technologies America, Inc.) as described previously (Fu et al., 2011 (link); Yee et al., 2012 (link)).