Biology subjects

Schenck, C.

Publications and source records attributed to Schenck, C..

7 recordsLinked to original sources

bioRxiv2026

Engineering plants tolerant to the toxic proline mimic azetidine-2-carboxylic acid through co-option of a bacterial detoxification mechanism

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

plant biology↗

bioRxiv2026

The proteotoxicity of azetidine 2-carboxylic acid is associated with reactive oxygen species accumulation

Plants make diverse metabolites to outcompete neighboring organisms for space and resources. Some of these toxic metabolites broadly disrupt conserved molecular mechanisms, such as protein biosynthesis. Nonproteogenic amino acids (NPAAs) are a structurally diverse class of metabolites that interfere with protein biosynthesis. The proline (Pro) analog azetidine-2-carboxylic acid (Aze) inhibits plant growth through misincorporation during protein biosynthesis. However, it is unknown if a cascade of downstream stress responses is triggered following Aze misincorporation. Here, we investigate the morphological and stress responses in Arabidopsis grown on Aze. Investigation of root morphological responses show not only reduced root growth, but increased root branching following growth on Aze. Altered root morphology is coupled with a reduced gravitropic response. Aboveground organs were also affected by Aze, including reduced chlorophyll content, reduced photosynthetic efficiency, and increased anthocyanin content. We then tested whether Aze induces reactive oxygen species (ROS) accumulation using multiple approaches and observed both immediate and sustained accumulation of general ROS and H2O2 following treatment with Aze. When plants were grown on Aze supplemented with Pro, ROS levels were restored to normal levels, suggesting that reducing misincorporation events results in less downstream stress responses. In summary, we find that following Aze treatment a cascade of downstream stress responses is induced that exacerbates the effects of toxic NPAAs. This study sheds light on the mechanism of action of NPAAs and provides information on the downstream consequences of translational errors.

plant biology↗

bioRxiv2025

A Metabolic Complex Involved in Tomato Specialized Metabolism

Specialized metabolites mediate diverse plant-environment interactions. Although, recent work has begun to enzymatically characterize entire plant specialized metabolic pathways, little is known about how different pathway components organize and interact within the cell. Here we use acylsugars - a class of specialized metabolites found across the Solanaceae family - as a model to explore cellular localization and metabolic complex formation of pathway enzymes. These compounds consist of a sugar core decorated with acyl groups, which are connected through ester linkages. In Solanum lycopersicum (tomato) four acylsugar acyltransferases (SlASAT1-4) sequentially add acyl chains to specific hydroxyl positions on a sucrose core leading to accumulation of tri and tetraacylated sucroses in the trichomes. To elucidate the spatial organization and interactions of tomato ASATs, we expressed SlASAT1-4 proteins fused with YFP in N. benthamiana and Arabidopsis protoplasts. Our findings revealed a distributed ASAT pathway with SlASAT1 and SlASAT3 localized to the mitochondria, SlASAT2 localized to the cytoplasm and nucleus, and SlASAT4 localized to the endoplasmic reticulum. To explore potential pairwise protein-protein interactions in acylsugar biosynthesis, we used various techniques, including co-immunoprecipitation, split luciferase assays, and bimolecular fluorescence complementation. These complementary approaches based on different interaction principles all demonstrated interactions among the different SlASAT pairs. Following transient expression of SlASAT1-4 in N. benthamiana, we were able to pull down a complex consisting of SlASAT1-4, which was confirmed through proteomics. Size exclusion chromatography of the SlASAT pulldown suggests a heteromultimeric complex consisting of SlASATs and perhaps other proteins involved in this interaction network. This study sheds light on the metabolic coordination for acylsugar biosynthesis through formation of an interaction network of four sequential steps coordinating efficient production of plant chemical defenses.

biochemistry↗

bioRxiv2024

Mode of action 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. However, lack of knowledge about defensive metabolite pathways, their modes of action, and resistance mechanisms limits our ability to manipulate these pathways for enhanced crop resilience. 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 mode of action of the NPAA azetidine-2-carboxylic acid (Aze), an analog of the amino acid proline (Pro). Using a combination of plate-based assays, metabolite feeding, metabolomics, and proteomics, 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 non-targeted proteomics confirms that Aze is misincorporated for Pro during protein biosynthesis, specifically on cytosolically translated proteins. qRT-PCR analysis and free amino acid profiling show that the unfolded protein response is upregulated during Aze treatment implicating protein degradation of misfolded proteins. This study demonstrates the mode of action of Aze in plants and provides a foundation for engineering Aze production and tolerance in crops for enhanced resilience.

plant biology↗

bioRxiv2023

Woolly mutation with Get02 locus overcomes the polygenic nature of trichome-based pest resistance in tomato

Type-IV glandular trichomes, which only occur in the juvenile phase of tomato development, produce acylsugars (AS) that broadly protect against arthropod herbivory. Previously, we introgressed the capacity to retain type-IV trichomes in the adult phase from Solanum galapagense into the cv. Micro-Tom (MT). The resulting MT-Get line contained five loci associated with enhancing the density of type-IV trichomes in adult plants. We genetically dissected MT-Get and obtained a sub-line containing only the locus on chromosome 2 (MT- Get02). This genotype displayed about half the density of type-IV trichomes compared to the wild progenitor. However, when we stacked the gain-of-function allele of WOOLLY, which codes for a HD-ZIP IV transcription factor, MT-Get02/Wo exhibited double the number of type-IV trichomes compared to S. galapagense. This discovery corroborates previous reports positioning WOOLLY as a master regulator of trichome development. AS levels in MT-Get02/Wo were comparable to the wild progenitor, although the composition of AS types differed, especially regarding less AS with medium-length acyl chains. Agronomical parameters of MT-Get02/Wo, including yield, were comparable to MT. Pest resistance assays showed enhanced protection against whitefly, caterpillar, and the fungus Septoria lycopersici. However, resistance levels did not reach that of the wild progenitor, suggesting the specificity of particulars AS types in the pest resistance mechanism. Our findings in trichome-mediated resistance advance the development of robust, naturally resistant tomato varieties, harnessing the potential of natural genetic variation. Moreover, by manipulating only two loci, we achieved exceptional results for a highly complex, polygenic trait, such as herbivory resistance in tomato.

plant biology↗

bioRxiv2022

Natural Variation Meets Synthetic Biology: Promiscuous Trichome Expressed Acyltransferases from Nicotiana acuminata

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely-related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. > 25 acylsugar variants could be produced in a single enzyme assay with four acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites. ONE SENTENCE SUMMARYAnalysis of Nicotiana glandular trichome metabolites and BAHD acyltransferases revealed diverse sucrose and glucose based acylesters.

biochemistry↗

bioRxiv2022

How to resolve conflicting conservation objectives: A decision support tool for the global selection of multi-purpose protected areas

The establishment and maintenance of protected areas (PAs) is viewed as a key action in delivering post-2020 biodiversity targets. PAs often need to meet multiple objectives, ranging from biodiversity protection to ecosystem service provision and climate change mitigation, but available land and conservation funding is limited. Therefore, optimizing resources by selecting the most beneficial PAs is vital. Here, we advocate for a flexible and transparent approach to selecting protected areas based on multiple objectives, and illustrate this with a decision support tool on a global scale. The tool allows weighting and prioritization of different conservation objectives according to user-specified preferences, as well as real-time comparison of the selected areas that result from such different priorities. We apply the tool across 1347 terrestrial PAs and highlight frequent trade-offs among different objectives, e.g., between species protection and ecosystem integrity. Outputs indicate that decision makers frequently face trade-offs among conflicting objectives. Nevertheless, we show that transparent decision-support tools can reveal synergies and trade-offs associated with PA selection, thereby helping to illuminate and resolve land-use conflicts embedded in divergent societal and political demands and values.

ecology↗