Redirecting tropane alkaloid metabolism reveals pyrrolidine alkaloid diversity in Atropa belladonna.

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

Effect of Nanopore Geometry in the Conformation and Vibrational Dynamics of a Highly Confined Molecular Glass.

The effect of nanoporous confinement on the glass transition temperature (Tg) strongly depends on the type of porous media. Here, we study the molecular origins of this effect in a molecular glass, N,N'-bis(3-methylphenyl)-N,N'-diphenylbenzidine (TPD), highly confined in concave and convex geometries. When confined in controlled pore glass (CPG) with convex pores, TPD's vibrational spectra remained unchanged and two Tg's were observed, consistent with previous studies. In contrast, when confined in silica nanoparticle packings with concave pores, the vibrational peaks were shifted due to more planar conformations and Tg increased, as the pore size was decreased. The strong Tg increases in concave pores indicate significantly slower relaxation dynamics compared to CPG. Given TPD's weak interaction with silica, these effects are entropic in nature and are due to conformational changes at molecular level. The results highlight the role of intramolecular degrees of freedom in the glass transition, which have not been extensively explored.

Specialized Metabolism in a Nonmodel Nightshade: Trichome Acylinositol Biosynthesis.

Plants make many biologically active, specialized metabolites, which vary in structure, biosynthesis, and the processes they influence. An increasing number of these compounds are documented to protect plants from insects, pathogens, or herbivores or to mediate interactions with beneficial organisms, including pollinators and nitrogen-fixing microbes. Acylsugars, one class of protective compounds, are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop naranjilla (lulo; Solanum quitoense) produces acylsugars containing a myoinositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the acylsucrose biosynthetic pathway of tomato (Solanum lycopersicum). Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin.

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway.

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored.