Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolism

Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action.