Comprehensive mass spectrometry‐guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae

Abstract

Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large-scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time-feasible manner. Here we introduce a workflow for large-scale, semi-automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well-known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co-occurring fragments and neutral losses, so-called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7-O-methyltransferase activity. Our workflow provides the foundations for the automated, high-throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms.

MolNetEnhancer: enhanced molecular networks by integrating metabolome mining and annotation tools

“…Another case where we demonstrate the efficiency of MolNetEnhancer for enhancing the chemical annotation of metabolomics data is our previous study on the plant family Rhamnaceae [25]. Rhamnaceae is a cosmopolitan family including about 900 species, and Rhamnaceae species are known for their exceptional morphological and genetic diversity, which are thought to be caused by the wide geographic distribution and different habitats [40].…”

“…After combining this chemical class annotations with taxonomic information of each molecular feature, the normalized distribution pattern of different classes of metabolites were analyzed. This revealed that the taxonomic clade Rhamnoid exhibits more diversified flavonoids, carbohydrate, and anthraquinones, while the Ziziphoid clade produces various triterpenoids and triterpenoid glycosides [25].…”

“…Publicly available mass spectrometry fragmentation data sets from four studies were used for this study. Details on how samples and data were collected can be found in the original studies [24,25,44,46]. Here, we list links to the different analyses that were done on each of the studies.…”

“…Furthermore, it would be practically advantageous to combine all these results in one place. We have previously described the integration of Mass2Motifs and chemical classifications with molecular networks to assess the chemical diversity within a subset of species of the plant genus Euphorbia [24] and the plant family Rhamnaceae [25]. However, in those studies, integration was achieved using custom in-house scripts in R, hampering adoption by the community.…”

Decomposing metabolite set activity levels with PALS

“…Molecular networking [31] uses spectral similarities to group metabolites into Molecular Families (MF). These MFs potentially correspond to chemical classes and have proven invaluable in enhancing the putative identifications of unknown metabolites [9,14]. In MS2LDA, metabolites are grouped by co-occurring fragments and neutral losses into Mass2Motifs that reveal potential chemical substructures [28].…”