Role of plant β-glucosidases in the dual defense system of iridoid glycosides and their hydrolyzing enzymes in Plantago lanceolata and Plantago major.

The typical defense compounds of Plantaginaceae are the iridoid glycosides, which retard growth and/or enhance mortality of non-adapted herbivores. In plants, glycosidic defense compounds and hydrolytic enzymes often form a dual defense system, in which the glycosides are activated by the enzymes to exert biological effects. Yet, little is known about the activating enzymes in iridoid glycoside-containing plants. To examine the role of plant-derived β-glucosidases in the dual defense system of two common plantain species, Plantago lanceolata and Plantago major, we determined the concentration of iridoid glycosides as well as the β-glucosidase activity in leaves of different age. To investigate the presence of other leaf metabolites potentially involved in plant defense, we used a metabolic fingerprinting approach with ultra-high performance liquid chromatography coupled with time-of-flight-mass spectrometry. According to the optimal defense hypothesis, more valuable parts such as young leaves should be better protected than less valuable parts. Therefore, we expected that both, the concentrations of defense compounds as well as the β-glucosidase activity, should be highest in younger leaves and decrease with increasing leaf age. Both species possessed β-glucosidase activity, which hydrolyzed aucubin, one of the two most abundant iridoid glycosides in both plant species, with high activity. In line with the optimal defense hypothesis, the β-glucosidase activity in both Plantago species as well as the concentration of defense-related metabolites such as iridoid glycosides correlated negatively to leaf age. When leaf extracts were incubated with bovine serum albumin and aucubin, SDS-PAGE revealed a protein-denaturing effect of the leaf extracts of both plantain species, suggesting that iridoid glycosides and plant β-glucosidase interact in a dual defense system.