Mechanism of action and biosynthesis of the toxic proline mimic azetidine 2-carboxylic acid in plants

Plants have an amazing capacity to outcompete neighboring organisms for space and resources. Toxic metabolites are major players in these interactions, which can have a broad range of effectiveness by targeting conserved molecular mechanisms, such as protein biosynthesis. Nonproteogenic amino acids (NPAAs) are a structurally diverse class of metabolites with a variety of functions but are typically not incorporated during protein biosynthesis. Here, we investigate the mechanism of action and biosynthesis of the NPAA azetidine-2-carboxylic acid (Aze), an analog of proline (Pro). We show that Aze inhibits the root growth of Arabidopsis and other plants. Aze-induced growth reduction was restored by supplementing L-, but not D-Pro, and nontargeted proteomics confirmed that Aze is misincorporated for Pro during protein biosynthesis, specifically on cytosolically translated proteins. Gene expression analysis, free amino acid profiling, and proteomics show that the unfolded protein response is upregulated during Aze treatment implicating that Aze misincorporation results in accumulation of misfolded proteins triggering a global stress response. To investigate how plants biosynthesize Aze, we screened over 80 diverse plants for accumulation of Aze. We find that Aze is narrowly distributed in some legumes and grasses and likely emerged through convergent evolution. To test the convergent evolution hypothesis and elucidate Aze biosynthesis, we assembled transcriptomes of two Aze producers. Crude enzyme assays and analysis of candidate genes reveal that Aze is biosynthesized from methionine by a transferase enzyme. To understand how plants that produce Aze can avoid autotoxicity. We focused on enzymes involved in protein biosynthesis and have resolved a structure via Cryo-EM with Aze bound of a Pro-tRNA synthetase that sheds light on how some plants can avoid Aze misincorporation. This study demonstrates the mechanism of action and biosynthesis of Aze in plants and provides a foundation for understanding the biological functions of proteotoxic metabolites.