A liverwort under stress: compound classification with CANOPUS to detect metabolic shifts

Liverworts are chemically diverse plants with unique cell organelles responsible for the synthesis and storage of specialized metabolites. Untargeted metabolomics was used to analyze the metabolic stress response of liverworts without isolating individual metabolites. CANOPUS classified the affected compounds, and helped to map the biochemical pathways of the unique stress response of liverworts compared to vascular plants.
Radula complanata
Radula complanata (Photo by Hermann Schachner on Wikimedia Commons)

The chemical diversity of liverworts 

Metabolites from bryophytes have received increased attention in recent years due to their potential cytotoxicity against human cancer cell lines​1​, herbicidal activity​2​, and fungicidal activity​3​. In particular one group of bryophytes, the liverworts, are incredibly chemically diverse despite their relatively simple physical morphology. They have unique cell organelles, so-called oil bodies, that are responsible for the synthesis and storage of most specialized metabolites produced by liverworts and are thought to be crucial for defense against herbivory​4​.

Untarget metabolomics

Special metabolites are often produced under stress. A group of researchers led by Kristian Peters from the Leibniz Institute of Plant Biochemistry studied the metabolic stress response of the leaf liverwort Radula complanata​5​. They exposed the plant to two growth-promoting hormones and three phytohormones for the stress response. They used untargeted metabolomics​6​ to analyze the full range of metabolic change without isolating individual metabolites.

The samples were separated using Bruker Elite HPLC coupled to a Bruker TIMS-TOF for measurement of MS1 spectra in positive and negative mode with electrospray ionization. Fragmentation spectra were measured in data-dependent acquisition mode, also referred to as automatic MS/MS. The researchers uploaded the set of spectra to MetaboLights​7​, a publicly available repository for metabolomics data.

Metabolite classification with CANOPUS

The researchers annotated the fragmentation spectra with SIRIUS​8​. First, they determined the molecular formulas with ZODIAC​9​, then predicted the molecular fingerprints with CSI:FingerID​10​ and used CANOPUS​11​ to assign the compound class based on the ClassyFire​12​ and NPClassifier​13​ ontologies. They only considered molecular formulas from natural product-based databases. In total, our software SIRIUS annotated 211 compound classes under the described conditions. The researchers illustrated the diversity of compound classes in a sunburst plot. An interactive, zoomable representation of the plot is available on Zenodo.

Metabolic stress response

Hormone treatments have only slightly affected the metabolism of R. complanata at a global level. Using statistical methods, the researchers examined the metabolic shifts. Of the 91 metabolites identified that fluctuate under the effects of phytohormones, 71 have been classified with CANOPUS. (Please note that CANOPUS actually returns a compound class for each submitted compound. Unclassified compounds might be due to missing or low quality fragmentation spectra.) Of these, 30 metabolites belong to primary metabolism and 27 to specialized metabolism (the remaining classes were too broad to be constrained to a specific metabolic type). Stress hormones largely downregulated primary metabolites and increased production of specialized metabolites. In contrast, growth hormones largely upregulated primary metabolite production and downregulated stress response metabolites. 

Sunburst plot showing an overview on the richness of classified metabolite compounds. (Picture from Blatt-Janmaat et al. 2023​5​ published under CC BY 4.0)

To map the functional relationships and biochemical pathways of these compounds, the researchers also created a molecular network. Most of the specialized metabolic pathways are involved in defense and are stimulated by stress hormone pathways. In liverworts, many of these specialized metabolites are found in the oil bodies, which are critical for herbivore defense, or have been identified as antifungal and are produced during fungal infections​3​. These results are consistent with stress responses of other moss species, but differ from results for vascular plants, highlighting the unique metabolic processes of liverworts. 

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The easy way to comprehensive structure elucidation​

SIRIUS is proven to be the best computational method for identifying molecules from tandem mass spectrometry data. SIRIUS is the umbrella application comprising molecular formula identification (ZODIAC), structure database search (CSI:FingerID), confidence score assignment (COSMIC) and compound class prediction (CANOPUS).​