Metabolomics Analysis Reveals Tissue-Specific Metabolite Compositions in Leaf Blade and Traps of Carnivorous Nepenthes Plants

Abstract

Nepenthes is a genus of carnivorous plants that evolved a pitfall trap, the pitcher, to catch and digest insect prey to obtain additional nutrients. Each pitcher is part of the whole leaf, together with a leaf blade. These two completely different parts of the same organ were studied separately in a non-targeted metabolomics approach in Nepenthes x ventrata, a robust natural hybrid. The first aim was the analysis and profiling of small (50–1000 m/z) polar and non-polar molecules to find a characteristic metabolite pattern for the particular tissues. Second, the impact of insect feeding on the metabolome of the pitcher and leaf blade was studied. Using UPLC-ESI-qTOF and cheminformatics, about 2000 features (MS/MS events) were detected in the two tissues. They showed a huge chemical diversity, harboring classes of chemical substances that significantly discriminate these tissues. Among the common constituents of N. x ventrata are phenolics, flavonoids and naphthoquinones, namely plumbagin, a characteristic compound for carnivorous Nepenthales, and many yet-unknown compounds. Upon insect feeding, only in pitchers in the polar compounds fraction, small but significant differences could be detected. By further integrating information with cheminformatics approaches, we provide and discuss evidence that the metabolite composition of the tissues can point to their function.

Transcriptomic and Proteomic Analyses of Nepenthes ampullaria and Nepenthes rafflesiana Reveal Parental Molecular Expression in the Pitchers of Their Hybrid, Nepenthes × hookeriana

“…These metabolites contain anti-microbial properties that prevent microbial competition for nutrient absorption. Efforts in the profiling of secondary metabolites from Nepenthes pitchers and their bioactivity are on-going ( Rosli et al, 2017 , 2018 ; Dávila-Lara et al, 2020 ). Meanwhile, genes involved in the biosynthesis of secondary metabolites such as phenylpropanoids, sesquiterpenoids, and triterpenoids in N. ampullaria were found to be influenced by endogenous protein depletion ( Goh et al, 2020 ).…”

Plumbagin, a Potent Naphthoquinone from Nepenthes Plants with Growth Inhibiting and Larvicidal Activities

“…As can be seen in Figure 2 A, starting at day 4 to 5, the presence of leaf but not pitcher tissue significantly affected the performance of the larvae, which gained less weight. At this point, it might be worth to mention that recently in N. x ventrata [ 24 ] and before in N. khasiana [ 15 ], the concentration of a NQ, very likely plumbagin, was determined to be significantly higher in leaves compared with pitchers, which may explain the result found in Figure 2 A. We also could support these results by comparing plumbagin content in pitcher vs. leaf; by quantitative NMR analysis, we found a 5.2-fold higher plumbagin concentration in leaf compared with pitcher tissue (650 and 125 µg g −1 FW, respectively).…”

“…Although significant, the growth inhibition effect was not very pronounced. Thus, the feeding experiment was repeated with dried leaf tissue in order to add more plant material to the diet, knowing that the water content of N. x ventrata tissue is about 90% [ 24 ]. Here, the effect of the plant tissue was more distinct ( Figure 2 B).…”

“…In addition to NQ, carotenoids, flavonoids, sterols, and triterpenes are described for Nepenthes leaves [ 1 , 22 , 23 ]. Recently, an untargeted metabolomics approach was performed in N. x ventrata comparing secondary metabolites of leaves and pitcher tissue before and after prey catches [ 24 ]. In that study, about 2000 compounds (MS/MS events) were detected in the two tissues showing enormous metabolome diversity, which was even higher in leaves.…”

“…Strikingly, the tissue specificity of chemical compounds could significantly discriminate pitchers from leaves. Besides many yet unknown compounds, the common constituents were phenolics, flavonoids, and NQ [ 24 ]. These data suggest that the metabolite composition of the tissues can point to their function.…”