α amylase ec 3

Extraction of leaf starch was performed as described in previous studies (Wanek et al., 2001 (link); Goettlicher et al., 2006 (link); Richter et al., 2009 (link)). Leaf starch was isolated from 50mg leaf material with methanol/chloroform/water (MCW, 12:5:3, v/v/v) at 70°C for 30min. Samples were centrifuged (10 000 ×g, 2min) and supernatants removed, while the leaf-starch-containing pellets were washed with MCW and deionized water and dried at room temperature (RT). Pellets were then re-suspended in water and boiled at 99°C for 15min to facilitate starch gelatinization. Subsequently, leaf starch was enzymatically digested with α-amylase (EC 3.2.1.1, Sigma-Aldrich, Buchs, Switzerland) at 85°C for 2h, and cleaned with centrifugation filters to remove enzymes (Vivaspin, Sartorius, Göttingen, Germany). To determine δ¹³C of bulk leaves (δ¹³C_leaf) and starch, an elemental analyser (Flash EA 1112 Series) coupled to a Delta^plusXP-IRMS was used (both Thermo Fisher, Bremen, Germany; Werner et al., 1999 (link)). Measurements of samples, blanks, and reference material followed the identical treatment principle described by Werner and Brand (2001) (link). The long-term precision of a quality control standard for all sequences was SD≤0.12‰.

All reagents used were of analytical grade, apart from methanol and acetonitrile which were of HPLC grade. Water was purified using Mili-Q system (Millipore; Vienna, Austria). Folate standards, folic acid (PteGlu), 5-methyltetrahydrofolate (5-CH₃-H₄PteGlu), 5-formyltetrahydrofolate (5-HCO-H₄PteGlu) and tetrahydrofolate (H₄PteGlu) were obtained from Sigma Aldrich (St. Louis, MO, USA); 10-formylfolic acid (10-HCO-H₄PteGlu) and 5,10-methenyltetrahydrofolate (5,10-CH⁺-H₄PteGlu) were obtained from Schircks Laboratories (Jona, Switzerland). Standards were all prepared as described by Konings [29 (link)]. 10-formyldihydrofolate (10-HCO-H₂PteGlu) was obtained from 5,10-methenyltetrahydrofolate according to Pfeiffer et al. [30 (link)]. Fresh rat plasma was purchased from Europa Bioproducts Ltd. (Cambridge, Great Britain), α-amylase (E.C.3.2.1.1) and protease (E.C.3.4.24.31) from Sigma Aldrich.