Exploiting Natural Variation of Secondary Metabolism Identifies a Gene Controlling the Glycosylation Diversity of Dihydroxybenzoic Acids in Arabidopsis thaliana

Abstract

Plant secondary metabolism is an active research area because of the unique and important roles the specialized metabolites have in the interaction of plants with their biotic and abiotic environment, the diversity and complexity of the compounds and their importance to human medicine. Thousands of natural accessions of Arabidopsis thaliana characterized with increasing genomic precision are available, providing new opportunities to explore the biochemical and genetic mechanisms affecting variation in secondary metabolism within this model species. In this study, we focused on four aromatic metabolites that were differentially accumulated among 96 Arabidopsis natural accessions as revealed by leaf metabolic profiling. Using UV, mass spectrometry, and NMR data, we identified these four compounds as different dihydroxybenzoic acid (DHBA) glycosides, namely 2,5-dihydroxybenzoic acid (gentisic acid) 5-O-b-D-glucoside, 2,3-dihydroxybenzoic acid 3-O-b-D-glucoside, 2,5-dihydroxybenzoic acid 5-O-b-D-xyloside, and 2,3-dihydroxybenzoic acid 3-O-b-D-xyloside. Quantitative trait locus (QTL) mapping using recombinant inbred lines generated from C24 and Col-0 revealed a major-effect QTL controlling the relative proportion of xylosides vs. glucosides. Association mapping identified markers linked to a gene encoding a UDP glycosyltransferase gene. Analysis of Transfer DNA (T-DNA) knockout lines verified that this gene is required for DHBA xylosylation in planta and recombinant protein was able to xylosylate DHBA in vitro. This study demonstrates that exploiting natural variation of secondary metabolism is a powerful approach for gene function discovery. P LANTS produce .200,000 diverse low-molecular weight compounds, known as secondary or specialized metabolites (Dixon and Strack 2003; Yonekura-Sakakibara and Saito 2009). These metabolites are not essential to shortterm survival but play important roles in many aspects of plant life, including growth regulation, defense against herbivores, UV protection, and other adaptations to the environment (Hartmann 2007). Apart from their roles in plant adaptation, plant specialized metabolites are rich sources for industrial and medicinal materials such as dyes, flavors, and pharmaceuticals (Balandrin et al. 1985).Studies of Arabidopsis thaliana have greatly advanced our understanding of the biochemical pathways and gene networks involved in a variety of specialized metabolism. For example, genetic analysis of artificially induced Arabidopsis mutants has led to the discovery of genes responsible for the biosynthesis of flavonoids, sinapate esters, and lignin (Shirley et al. 1995;Ruegger and Chapple 2001). Exploiting natural variation between different accessions in the accumulation of glucosinolates and terpenoids has led to the identification of genes involved in their biosynthesis and alleles regulating their variation that are under strong selection (Kliebenstein et al. 2001a,b;Kroymann et al. 2001;Chen et al. 2003;Tholl et al. 2005). Despite this progress, there are many mo...

Discovering variation of secondary metabolite diversity and its relationship with disease resistance inCornus floridaL.

“…The abundance and inducibility of certain secondary metabolites such as flavonoids, other phenolics, and glycoside derivatives have been found to be heritable and mediated by herbivore–pathogen pressures (Johnson, Agrawal, Maron, & Salminen, 2009; Li et al., 2014). Determining the specific genetic factors regulating such adaptive metabolites remains an important goal, and this study adds to emerging efforts to integrate large secondary metabolite concentration data with information from genome scans (Eckert et al., 2012; Jensen, Foll, & Bernatchez, 2016; Talbot et al., 2016).…”

“…This model has been used in genomewide association (GWA) studies of Arabidopsis thaliana (Bac‐Molenaar, Fradin, Rienstra, Vreugdenhil, & Keurentjes, 2015; Fournier‐Level et al., 2011; Li, Huang, Bergelson, Nordborg, & Borevitz, 2010; Li et al., 2014; Strauch et al., 2015) because of its computational efficiency, and its ability to handle and control for population stratification (Price, Zaitlen, Reich, & Patterson, 2010) and environment. We tested for both SNP associations to each metabolite and for SNP associations to the property of chemical richness.…”

S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis

“…Similar to other hydroxybenzoates, 2,5-DHBA accumulates as glycoconjugates in plants, primarily as 2,5-DHBA 5-O-b-D-glucosides, 2,5-DHBA 5-O-b-D-xylosides, or 2,5-DHBA 2-O-b-D-xylosides (Dean and Delaney, 2008;Tárraga et al, 2010;Li et al, 2014). 2,5-DHBA accumulates in response to different types of plant-pathogen interactions in much higher levels than SA (Bellés et al, 1999(Bellés et al, , 2006Campos et al, 2014).…”

“…Moreover, 2,5-DHBA itself displays antibacterial activity in the harvested fruits (Lattanzio et al, 1996). Although the enzymes UGT89A2 and GAGT, which are responsible for the conversion of 2,5-DHBA to its glycosides, have been well characterized (Lim et al, 2002;Tárraga et al, 2010;Li et al, 2014) and the in vivo feeding of a 14 C-labeled SA tracer in Gaultheria procumbens suggested that 2,5-DHBA was formed from the substrate SA half a century ago (Ibrahim and Towers, 1959), the enzymes responsible for the formation of 2,5-DHBA are still a mystery.…”

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

“…Several studies of Arabidopsis natural accessions (individuals collected from wild populations) revealed considerable qualitative and quantitative variation in the accumulation of various compounds such as glucosinolates, terpenoids, and phenylpropanoids (2)(3)(4). This extensive metabolite variation can be attributed to genetic variation in genes encoding enzymes and regulatory factors of the pathways involved; quantitative trait locus (QTL) mapping has successfully uncovered several genes involved in the production of these metabolites (3)(4)(5)(6)(7). Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolic profiling has further extended such analysis to unknown metabolites, finding genetic contribution to the variation in at least three-fourths of detected mass peaks (8).…”