Figure 1.

Screen of 18 Arabidopsis UGTs for their ability to glycosylate NHP. A, Biosynthetic pathway for the production of NHP-Glc from l-Lys in Arabidopsis. The biosynthetic activity of UGT76B1 was characterized in this work. B, Abundance of NHP-Glc measured with LC–MS after transient expression of GFP or respective Arabidopsis UGTs alongside Arabidopsis ALD1 + FMO1 in N. benthamiana leaves. In the initial screen, Agrobacterium strains harboring distinct UGTs were combined in equal proportions and co-infiltrated with Agrobacterium strains harboring ALD1 and FMO1. In the second screen, Agrobacterium strains harboring UGT76B1, UGT76F2, or UGT85A1 were separately co-infiltrated with Agrobacterium strains harboring ALD1 and FMO1. Total inoculum (OD₆₀₀) was kept constant in both experiments by including Agrobacteria harboring GFP as a control. Bars represent the mean ± sd (n = 3 independent biological replicates). Values reported as zero indicate no detection of metabolites. Asterisks indicate significant differences in NHP-Glc levels (one-tailed t test; *P < 0.05, **P < 0.01). The experiment was repeated two times with similar results. C, Representative LC–MS chromatograms of NHP-Glc (m/z = 308.134) in extracts from N. benthamiana (black) and Arabidopsis adult leaves (blue) transiently expressing ALD1 + FMO1 + UGT76B1 after infiltration with 1 mM NHP synthetic standard. D, Comparative MS/MS spectra of NHP-Glc in extracts from N. benthamiana (black) and Arabidopsis adult leaves (blue) transiently expressing ALD1 + FMO1 + UGT76B1 after infiltration with 1 mM NHP synthetic standard at collision energies of 10 and 40 V.