Abstract
Nepenthes is a genus comprising carnivorous tropical pitcher plants that have evolved trapping organs at the tip of their leaves for nutrient acquisition from insect trapping. Recent studies have applied proteomics approaches to identify proteins in the pitcher fluids for better understanding the carnivory mechanism, but protein identification is hindered by limited species-specific transcriptomes for Nepenthes. In this study, the proteomics informed by transcriptomics (PIT) approach was utilized to identify and compare proteins in the pitcher fluids of Nepenthes ampullaria, Nepenthes rafflesiana, and their hybrid Nepenthes × hookeriana through PacBio isoform sequencing (Iso-Seq) and liquid chromatography-mass spectrometry (LC-MS) proteomic profiling. We generated full-length transcriptomes from all three species of 80,791 consensus isoforms with an average length of 1,692 bp as a reference for protein identification. The comparative analysis found that transcripts and proteins identified in the hybrid N. × hookeriana were more resembling N. rafflesiana, both of which are insectivorous compared with omnivorous N. ampullaria that can derive nutrients from leaf litters. Previously reported hydrolytic proteins were detected, including proteases, glucanases, chitinases, phosphatases, nucleases, peroxidases, lipid transfer protein, thaumatin-like protein, pathogenesis-related protein, and disease resistance proteins. Many new proteins with diverse predicted functions were also identified, such as amylase, invertase, catalase, kinases, ligases, synthases, esterases, transferases, transporters, and transcription factors. Despite the discovery of a few unique enzymes in N. ampullaria, we found no strong evidence of adaptive evolution to produce endogenous enzymes for the breakdown of leaf litter. A more complete picture of digestive fluid protein composition in this study provides important insights on the molecular physiology of pitchers and carnivory mechanism of Nepenthes species with distinct dietary habits.
Highlights
Nepenthes comprises unique carnivorous tropical plants with pitcher organs at the ends of leaf tips for the capture, digestion, and absorption of insects to grow in nutrient-poor soil
Transcriptomics and proteomics analyses of N. ampullaria, N. rafflesiana, and their hybrid, N. × hookeriana were conducted with an overview of the methods illustrated in Supplementary Figure 1
Despite having the lowest number of read of insert (ROI), the hybrid expressed the highest number of consensus isoforms, indicating more varied transcripts in the hybrid
Summary
Nepenthes comprises unique carnivorous tropical plants with pitcher organs at the ends of leaf tips for the capture, digestion, and absorption of insects to grow in nutrient-poor soil. Nepenthes species are mostly insectivorous but previous studies showed that Nepenthes ampullaria, which is predominantly found in the heath and swamp forests compared to open habitats like other species, possesses detritivore traits to trap leaf litter as a nutrient source (Moran et al, 2003). This genus is well-known for natural and artificial hybridization. There is no comprehensive report comparing the molecular expression in the pitchers and pitcher fluids of the hybrid and N. rafflesiana as carnivores, with N. ampullaria being an omnivore This gap of knowledge was pointed out by Pavlovic (2012) on the adaptive radiation of Nepenthes nutrient sequestration strategies
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