Enzyme-Linked Assays of Metabolites
Enzymatic assays were carried out on extracts from fully expanded leaves
as described previously (Dyson et al. , 2016). Starch, sucrose and
glucose, and malate were measured using total starch (Method E), sucrose
D-glucose, and l-malic acid assay kits (Megazyme), respectively. To
measure fumarate, the malate kit was modified to include an extra
independent reaction step, using 2 units of fumarate hydratase enzyme
(Sigma). Data were analysed using an analysis of variance, followed by a
Tukey post-hoc test, in SPSS (IBM).