Metabolic consequences of knocking outUGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin inSorghum bicolor(L. Moench)

Abstract

Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

“…3b). Functionally characterized New Phytologist members of group G are involved in the biosynthesis of the iridoid geniposide (GjUGT6) in gardenia (Nagatoshi et al, 2011), 7-deoxyloganic acid (CrUGT8; UGT709C2) in Catharanthus roseus (Asada et al, 2013), different terpenes (VvGT14) in V. vinifera (Bonisch et al, 2014) and Camellia sinensis (UGT85K11) (Ohgami et al, 2015), cyanogenic glucosides (UGT85B1) in Sorghum bicolor (Hansen et al, 2003;Blomstedt et al, 2016), and cytokinins (UGT85A1) in Arabidopsis thaliana (Jin et al, 2013). Amino acid sequence comparisons showed 43% and 40% sequence identity with CrUGT8 and GjUGT6, respectively, which are involved in the glycosylation of monoterpenoid-related secondary metabolites (Fig.…”

Micro‐ and macroevolutionary adaptation through repeated loss of a complete metabolic pathway

“…For CNglcs specifically and plant chemical defense metabolites more generally, the toxicity of intermediates in the biosynthetic pathway is likely a critical factor driving physical linkage and coinheritance of the component genes (Kristensen et al, 2005;Mylona et al, 2008;Blomstedt et al, 2016;. In the case of CNglcs, the oximes and cyanohydrins that are produced as intermediates during their synthesis are highly unstable and reactive (Gleadow & Møller, 2014).…”

Plant Desiccation Tolerance and its Regulation in the Foliage of Resurrection “Flowering-Plant” Species

“…Another powerful functional genetics methodology, which does not necessitate the development of DNA transformation technology, involves targeting induced local lesions in genomes (TILLING) [171,172]. This approach has been successfully used to identify mutants in monocotyledonous crops that possess disruptions in specific genes [172][173][174]. An alternative approach which has proven valuable and which may be of direct interest to agricultural crops, is the ectopic expression of genes from resurrection plants in otherwise desiccation-sensitive species where reproducible transformation protocols do exist.…”