Uncovering the genetic and molecular mechanisms driving evolution and adaptation to ever-changing environments is a grand challenge in biology, with implications for creating a stable supply of food, fiber, industrial feedstock and pharmaceutical products from plants. Plants grow in diverse ecological niches and collectively must respond to environmental fluctuations ranging from seconds to centuries. Such diverse evolutionary histories are associated with adaptations that are found in specific plant groups, including production of metabolites that protect plants from abiotic stress, reduce herbivory and pathogenic bacteria and fungi, and attract beneficial animals (e.g. pollinators) and symbiotic microbes (famously, soil bacteria involved in converting atmospheric nitrogen to fertilizer in legumes). Plants collectively produce hundreds of thousands of these lineage-specific specialized metabolites in specific cells or tissues, at distinct developmental stages, or in response to environmental stimuli.

Scientific Merit
This project focused on two general types of specialized metabolites found in the 'nightshade family' (Solanaceae), which represent the diversity of structures and activities of these metabolites.

Tropane and pyrrolidine alkaloids are medicinally important plant metabolites that are synthesized in several plant families, including the Solanaceae. Several hundred tropane and pyrrolidine alkaloids are known but not all members of the Solanaceae synthesize these metabolites. In this project we focused on developing Atropa belladonna (Deadly Nightshade) to study the biosynthesis of these alkaloids. Using a combination of metabolomics, gene silencing, and biochemical characterization, we identified enzymes required for the biosynthesis of hyoscyamine and scopolamine, two medicinally important metabolites. This research enabled other scientists to engineer the production of these metabolites in yeast. We also developed a discovery metabolomics pipeline, which when combined with gene-silencing led to the identification of several novel alkaloids and enzymes required for early steps in the biosynthesis of calystegines, a subclass of tropane alkaloids. A similar strategy was utilized to reveal the existence of a novel pyrrolidine alkaloid metabolic subnetwork from A. belladonna comprising several dozen previously unreported metabolites. The in planta roles of the novel alkaloids identified through this project await discovery but these metabolites may possess utility as pharmaceuticals, antimicrobials, or show efficacy against insect pests.  

Acylsugars protect Solanaceae plants from insect herbivores and fungal pathogens. In this project we demonstrated a surprisingly wide range of structures produced by trichomes of domesticated crop plants (cultivated tomato or Solanum lycopersicum, eggplant or Solanum melongena) and wild species (for example, black nightshade or Solanum nigrum). Genomic, gene editing and biochemical methods were used to discover enzymes that produce these compounds and we revealed fascinating evolutionary mechanisms that produce these structurally diverse compounds. For example, we found strong evidence that glucose-based acylsugar biosynthesis evolved independently twice by independently 'borrowing' and modifying the function of the invertase enzyme of carbohydrate metabolism. The production of acylsugars in tomato trichomes was modified by CRISPR-Cas gene editing, and we found that these modifications resulted in changes to feeding behavior of hornworm herbivores.   

Beyond the classes of specialized metabolites we focused on, the biosynthetic pathways of most specialized metabolites are unknown. In addition, there are an abundance of metabolic enzyme genes in plant genomes with unknown roles in primary or specialized metabolism. With the experimental data generated from this project and earlier studies, we established computational models capable of distinguishing enzyme genes involved in primary and specialized metabolism in the model plant Arabidopsis. We also demonstrated that such prediction models could be applied across species with a proof-of-concept study in tomatoes. Finally, we show that tomato biosynthetic pathways can be predicted by judicious use of gene expression data. 

Broader impacts
The broader impacts of this project included workforce development and scientific innovation. Dozens of undergraduate students from Michigan State and colleges and universities across the U.S. were involved in the project. Eight graduate students were trained in computational and experimental approaches, 10 postdoctoral researchers participated in the research and trained earlier career participants. Through this project, we have also engaged the public through Michigan State University Science Fair, Girls' Math and Science Day, Darwin Day, area public libraries, and high schools by running exhibits, educational games, and/or presentations.

Last Modified: 09/13/2022
Modified by: Robert L Last