Using the knowns to discover the unknowns: MS‐based dereplication uncovers structural diversity in 17‐hydroxygeranyllinalool diterpene glycoside production in the Solanaceae

Abstract

SUMMARYExploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometrybased metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross-species comparisons. 17-hydroxygeranyllinalool diterpene glycosides (HGLDTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL-DTGs result in extensive in-source fragmentation (IS-CID) during ionization. To reconstruct these IS-CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive-ion spectra of purified HGL-DTGs. From this library, 251 nonredundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL-DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL-DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS-based workflow is readily applicable for cross-species re-identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.

JA but not JA‐Ile is the cell‐nonautonomous signal activating JA mediated systemic defenses to herbivory in Nicotiana attenuata

“…We determined the local and systemic accumulations of OS‐elicited 17‐hydroxygeranyllinalool diterpene glycosides (DTGs) which negatively influence larval performance as an anti‐feedant (Lou and Baldwin ; Jassbi et al ; Heiling et al ; Heiling et al ). Levels of total DTGs were significantly increased in ir AOC scions grafted onto WT stocks compared to scions of ir AOC self‐grafted plants after W + OS treatment (Figure B; P < 0.001).…”

“…Both JA deficient lines demonstrated different patterns of accumulations of individual DTGs (Figure ; P < 0.001). For this analysis, we quantified four classes of DTGs: the precursor with glucosyl (lyciumoside I), core compounds with glucosyl and rhamnosyl, but lacking malonyl groups (lyciumoside IV, nicotianoside III, and attenoside), singly malonylated compounds with glucosyl and rhamnosyl (nicotianosides I, IV, and VI) and dimalonylated compounds with glucosyl and rhamnosyl (nicotianosides II, V, and VII) (Heiling et al ; Heiling et al ). The precursor lyciumoside I accumulated to higher levels in the scions of self‐grafted JA‐deficient genotypes compared to WT/WT after OS‐elicitation (Figure , P < 0.001).…”

Modern mass spectrometry for synthetic biology and structure-based discovery of natural products

“…61 Going beyond simple dereplication (and microbial sources), Heiling et al compared the MS 2 spectra of known 17-hydroxygeranyllinalool diterpene glycosides to unknown metabolites and were able to propose an additional 105 novel structural analogues from 35 species of plants. 62 …”

First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum

“…In addition to lysine acetylation, some new types of PTMs, such as malonylation and lysine succinylation, were identified using mass spectrometry combined with the affinity purification of modified peptides using antibodies directed against these modifications 14–20 . Similar to lysine acetylation, lysine malonylation and succinylation are important in regulating cellular metabolism, and both processes exist in eukaryotes and prokaryotes 21–24 .…”