Saint-Vincent Patricia M B, Furches Anna, Galanie Stephanie, Teixeira Prates Erica, Aldridge Jessa L, Labbe Audrey, Zhao Nan, Martin Madhavi Z, Ranjan Priya, Jones Piet, Kainer David, Kalluri Udaya C, Chen Jin-Gui, Muchero Wellington, Jacobson Daniel A, Tschaplinski Timothy J
Center for Bioenergy Innovation, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States.
Bredesen Center for Interdisciplinary Research, University of Tennessee, Knoxville, TN, United States.
Front Plant Sci. 2023 Jul 21;14:1210146. doi: 10.3389/fpls.2023.1210146. eCollection 2023.
Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as , a biofuel feedstock and model woody plant species. Despite their growing importance in plant genetics and metabolomics, few mGWASs are experimentally validated. Here, we present a functional genomics workflow for validating mGWAS-predicted enzyme-substrate relationships. We focus on uridine diphosphate-glycosyltransferases (UGTs), a large family of enzymes that catalyze sugar transfer to a variety of plant secondary metabolites involved in defense, signaling, and lignification. Glycosylation influences physiological roles, localization within cells and tissues, and metabolic fates of these metabolites. UGTs have substantially expanded in , presenting a challenge for large-scale characterization. Using a high-throughput assay, we produced substrate acceptance profiles for 40 previously uncharacterized candidate enzymes. Assays confirmed 10 of 13 leaf mGWAS associations, and a focused metabolite screen demonstrated varying levels of substrate specificity among UGTs. A substrate binding model case study of UGT-23 rationalized observed enzyme activities and mGWAS associations, including glycosylation of trichocarpinene to produce trichocarpin, a major higher-order salicylate in We identified UGTs putatively involved in lignan, flavonoid, salicylate, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress response that determine sustainable biomass crop production. Our results provide new support for analyses and evidence-based guidance for functional characterization.
代谢物全基因组关联研究（mGWAS）越来越多地用于发现植物中目标表型的遗传基础，如作为生物燃料原料和模式木本植物物种的 。尽管它们在植物遗传学和代谢组学中的重要性日益增加，但很少有mGWAS得到实验验证。在这里，我们提出了一个功能基因组学工作流程，用于验证mGWAS预测的酶-底物关系。我们专注于尿苷二磷酸糖基转移酶（UGT），这是一类催化糖转移到参与防御、信号传导和木质化的多种植物次生代谢物的酶。糖基化影响这些代谢物的生理作用、在细胞和组织中的定位以及代谢命运。UGT在 中大量扩增，这给大规模表征带来了挑战。使用高通量分析，我们生成了40种先前未表征的候选酶的底物接受谱。分析证实了13个叶片mGWAS关联中的10个，并且一个聚焦的代谢物筛选显示UGT之间具有不同水平的底物特异性。UGT-23的底物结合模型案例研究使观察到的酶活性和mGWAS关联合理化，包括将毛果芸香烯糖基化以产生毛果芸香苷，这是 在 中的一种主要高阶水杨酸盐。我们鉴定出可能参与木脂素、黄酮类、水杨酸盐和植物激素代谢的UGT，这对决定可持续生物质作物生产的细胞壁生物合成、氮吸收以及生物和非生物胁迫反应具有潜在影响。我们的结果为 分析提供了新支持，并为 功能表征提供了基于证据的指导。
