Functional Characterization of Genes Coding for Novel -D-Glucosidases Involved in the Initial Step of Secoiridoid Glucosides Catabolism in Rafn.

Secoiridoid glucosides (SGs) are monoterpenoids derived from the iridoid cyclopentane-C-pyran skeleton with -D glucose linked at C1 position. Coordinated metabolic processes, such as biosynthesis and catabolism of SGs, ensure constitutive presence of these bitter tasting compounds in plant tissues, which plays a decisive role in the defense against pathogens and herbivores. These compounds are susceptible to hydrolysis mediated by enzymes -glucosidases, and the resulting aglycones are subsequently directed toward different metabolic pathways in plants. Function of two -D-glucosidases (named BGlu1 and BGlu2) from centaury ( Rafn; fam. Gentianaceae), belonging to the glycoside hydrolase 1 (GH1) family, was confirmed using assays with recombinant proteins, following their heterologous expression in and His-tag affinity purification. Although they show slightly differential substrate preference, both isoforms display high specificity toward SGs and the organ-specific distribution of transcripts was positively correlated with the content of SGs in diploid and tetraploid plants. Transient overexpression of BGlu1 and BGlu2 in leaves induced changes in metabolite profiles. The effectiveness of transgene overexpression has been altered by plant ploidy. UHPLC/DAD/(±)HESI - MS profiling of leaves of diploid and tetraploid genotypes revealed that the amounts of major SGs; sweroside, swertiamarin, and gentiopicrin was decreased in agroinfiltrated leaves, especially when BGlu1 and BGlu2 were co-expressed with transgene silencing suppressor p19. The work demonstrates that metabolic engineering adopting transient overexpression of BGlu1 and BGlu2 is a suitable tool for the modulation of SGs content and glucosides/aglycones ratio, which might have substantial effects on overall phytochemistry of .