UPLC-TOF-MS/MS-Based Metabolomics Analysis Reveals Species-Specific Metabolite Compositions in Pitchers of Nepenthes ampullaria, Nepenthes rafflesiana, and Their Hybrid Nepenthes × hookeriana

Abstract

Hybridization is key to the evolution and diversity of plants in nature. Nepenthaceae comprises a family of diverse tropical carnivorous pitcher plant species with extensive hybridization. However, there is no study to date on the metabolite expression of hybrids in this family. We performed a non-targeted metabolomics analysis of the pitchers of two Nepenthes species with different dietary habits, namely, the semi-detritivorous N. ampullaria and carnivorous N. rafflesiana with their hybrid (N. × hookeriana) for a comparative study. The whole-pitcher samples were extracted in methanol:chloroform:water (3:1:1) via sonication-assisted extraction and analyzed using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) followed by data analysis to profile chemical compositions. A total of 1,441 metabolite features were profiled from the three species in which 43.3% of features in the hybrid samples were not found in either of its parents. The partial least squares discriminant analysis (PLS-DA) found 324 metabolite features with variable in projection (VIP) values greater than one in which 55 features were statistically significant. This showed that the hybrid is closer to N. rafflesiana, which is consistent to the previous study on gene and protein expressions. A total of 105 metabolites were putatively identified with manual searches using public metabolite databases. Phenols were detected to be the most abundant secondary metabolites due to a high flavonoid content, especially in N. rafflesiana. The most abundant feature 476.3s:449.102 was found to be the most significant VIP for distinguishing between the three species as a chemical marker. This is the first study comparing metabolites in the carnivory organs of different Nepenthes species with comprehensive profiling and putative identification. The differential metabolite compositions in the pitchers of different species might have ecological implications with the hybrid showing intermediate phenotype between the parents as well as manifesting unique metabolites. However, there is no clear evidence of metabolites related to the differences in dietary habits between the hybrid and the two parent species.

Neprosin belongs to a new family of glutamic peptidase based on in silico evidence

“…Proteases have been reported to be secreted into the pitcher fluids of Nepenthes, which is a genus in the Nepenthaceae tropical carnivorous pitcher plant family with over 170 species that are studied to understand the physiology and mechanisms of botanical carnivory (Clarke et al 2018). With high-throughput technology, omics-based explorations have been conducted on the Nepenthes pitcher tissues and fluids for biomolecular discovery and molecular physiology studies based on transcriptomics, proteomics, and metabolomics approaches (Ravee et al 2021;Rosli et al 2021). Proteases in the pitcher fluids have unique characteristics such as higher enzyme stability in a wider range of temperature and pH conditions (Ravee et al 2018).…”

Comparative metabolomics analysis reveals alkaloid repertoires in young and mature Mitragyna speciosa (Korth.) Havil. Leaves

“…MS/MS analysis for young and mature leaf samples of M . speciosa was done according to Rosli et al [ 62 ] by pooling replicates of all extracts at equal amounts. Tandem mass spectra were acquired in Auto-MS/MS and multiple reaction monitoring (MRM) mode to facilitate compound identification.…”

Omics Approaches in Uncovering Molecular Evolution and Physiology of Botanical Carnivory

“…The comparative analysis determined that transcripts and proteins identified in the hybrid N. × hookeriana resemble more of the insectivorous N. rafflesiana than the omnivorous N. ampullaria , which can derive nutrients from leaf litter. A metabolomics approach has been taken to study the effect of hybridization between the two aforementioned Nepenthes species with a consistent conclusion based on their chemical compositions and the putative identification of a flavonoid, astragalin as a distinctive molecular marker among the three species [ 22 , 35 ].…”