Amyloglucosidase

Soluble sugars and starch were determined in fully expanded leaves according to Kim et al. (2013 (link)). 50 mg frozen flag leaf material were homogenized in liquid nitrogen, dissolved in 0.75 ml of 80% (v/v) ethanol and incubated at 80°C for 60 min. Crude extracts were centrifuged at 14,000 rpm for 5 min at 4°C, and the upper phase was concentrated in a speed vacuum concentrator (Christ, Germany) at 45°C for 180 min. The pellet was resuspended in 0.25 ml HPLC-grade water and shaken for 15 min at 4°C. Hexokinase (HK), phosphoglucoisomerase (PGI) and β-fructosidase were added successively to measure Glc, Fru and Suc as described in Kim et al. (2013 (link)). The residue of sugar extraction was washed twice with 1 ml of 80% (v/v) ethanol. Starch was decomposed with 0.4 ml of 0.2 N KOH for 16 h at 4°C and neutralized with 70 μl of 1 M acetic acid. Hydrolysis of starch was performed using a 1:1 ratio of sample and a buffer containing 50 mM sodium acetate, pH 5.2 and 7 units mg⁻¹ of amyloglucosidase (Roche, Germany). The cocktail was incubated for 16 h at 37°C. Determination of produced Glc was performed according to Kim et al. (2013 (link)).
Free amino acids were extracted and determined as described by Höller et al. (2014 (link)). Analysis and quantification of metabolites were performed essentially as described by Hosseini et al. (2016 (link)).

The total starch (TS) and resistant starch (RS) contents were determined using the methodology reported by Goñi et al. [28 (link)]. For the determination of the TS, an enzymatic solution of amyloglucosidase (brand Roche, No. 102,857, Roche Diagnostics, Indianapolis, IN, USA) was used. The RS content was quantified using different enzyme solutions and incubation times as follows, 60 min incubation with pepsin, 16 h incubation with α-amylase, and 45 min incubation with amyloglucosidase. The glucose released by enzymatic digestion was quantified using the glucose oxidase/peroxidase assay (SERA-PAK^® Plus, Bayer de Mexico, SA de CV), reading the optical densities of the samples at 510 nm. The percentage of starch hydrolysis (the rate of in vitro digestion) was determined according to the methodology reported by Zamudio-Flores et al. [29 ].

The harvested plants were quickly freeze-dried, and the different organs (leaves, stems and roots) were weighed and ground into powder. The glucose, fructose and sucrose contents in the organs were measured according to the enzymatic method [20 (link)]. The starch contents in the organs were assessed by a method using amyloglucosidase and α-amylase (Roche Diagnostics GmbH, Mannheim, Germany). Starch was not detected in these organs, indicating an absence of starch in this species. Therefore, starch was not measured in the experiment. Next, another important polysaccharide, fructan, was measured using an enzymatic Fructan Assay Kit (Megazyme Ltd., I Wicklow, reland), and a high amount of fructans, instead of starch, was observed in the plants. Thus, the fructan content in various plant parts was analyzed using the same Fructan Assay Kit.