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

Abstract

Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. As an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolitegene associations led to the identification of N-malonyl-D-alloisoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5′-phosphate for its activity. This study thus identifies a new D-amino acid racemase gene family and advances our knowledge of plant Damino acid metabolism that is currently largely unexplored. It also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism.D-amino acid | racemase | genome-wide association | secondary metabolism | natural variation P lants have the ability to create over 200,000 small compounds known as secondary or specialized metabolites (1). These chemically diverse compounds help mediate plant adaptation to their environment and play important roles in plant defense mechanisms, pigmentation, and development. In addition, many of these metabolites are desirable to humans as medicinal and nutritional compounds. Therefore, furthering our understanding of plant specialized metabolism will have profound impacts on various applications from crop improvement to human health.To date, only a small fraction of the chemical and catalytic space in plant specialized metabolism has been explored. Even in the best-studied model plant Arabidopsis thaliana, there are still many uncharacterized metabolites, and the vast majority of genes encoding enzymes implied to be involved in specialized metabolism do not have known associations with any metabolites. 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 (2-4). 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 uncove...

A Guide to Genome‐Wide Association Mapping in Plants

“…The second reason for thinking carefully about phenotype is because the probability of GWAS succeeding is directly related to the number and effect sizes of genes responsible for variation: The fewer the genes and the larger the effects, the more power for identifying the genes. Consistent with the expectation that it is easier to identify loci underlying variation in traits with a simple genetic basis, some of the strongest statistical signals are for metabolic traits for which there is a single enzyme or transporter that is responsible for distinct phenotypes (Lipka et al., ; Chen et al., ; Matsuda et al., ; Strauch et al., ; Shakoor et al., ). By contrast, we should not expect GWAS to provide extremely strong signals for genes that contribute to variation in highly complex multigenic quantitative traits such as height.…”

Genetic Components of Root Architecture Remodeling in Response to Salt Stress

“…While many GWAS studies published to date focus on single traits, such as ion accumulation, main root growth, or compatible solute accumulation (Strauch et al, 2015;Baxter et al, 2010;Lachowiec et al, 2015;Slovak et al, 2014;Verslues et al, 2014), there is an increasing focus on multitrait response phenotypes, including RSA response to stress (Rosas et al, 2013;Kawa et al, 2016). An advantage of performing GWAS on multitrait phenotypes, also apparent in our study, is that additional confidence can be gained when the same candidate loci are mapped using different traits and/or different stress conditions or when the multitrait phenotypes are reduced to principle components that map to the overlapping loci ( Figure 3A).…”

Discovering variation of secondary metabolite diversity and its relationship with disease resistance in Cornus florida L.

“…Metabolites were extracted following a modified protocol of Strauch, Svedin, Dilkes, Chapple, and Li (2015). For each tree, extraction material (20 mg) was selected from all sampled leaf tissue visibly free of mold or necrosis to minimize the chances of sampling metabolites that were unique to fungi, altered or degraded during collection.…”

“…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.…”