SUMMARYManihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from L-valine and L-isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside-specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2-hydroxy-2-methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co-occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co-expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine-tuning nitrogen assimilation in cassava.

SUMMARYIn comparison with the technology platforms developed to localize transcripts and proteins, imaging tools for visualization of metabolite distributions in plant tissues are less well developed and lack versatility. This hampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon may be used to accurately image the distribution of even labile plant metabolites such as hydroxynitrile glucosides, which normally undergo enzymatic hydrolysis by specific b-glucosidases upon cell disruption. This fast and simple sample preparation resulted in no substantial differences in the distribution and ratios of all hydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a b-glucosidase mutant plant that lacks the ability to hydrolyze certain hydroxynitrile glucosides. In wild-type, the enzymatic conversion of hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restricted area of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme in hydroxynitrile glucoside biosynthesis in L. japonicus leaves, CYP79D3, was found to be highly expressed during the early stages of leaf development, and the hydroxynitrile glucoside distribution in mature leaves reflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated using cryo-sections of cassava (Manihot esculenta) tubers. The hydroxynitrile glucoside levels were highest in the outer cell layers, as verified by LC-MS analyses. The unexpected discovery of a hydroxynitrile-derived di-glycoside shows the potential of DESI-MSI to discover and guide investigations into new metabolic routes.

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