Transcriptome-wide shift from photosynthesis and energy metabolism upon endogenous fluid protein depletion in young Nepenthes ampullaria pitchers

Abstract

faris 'imadi Mohd Salleh, Rishiesvari Ravee, Wan nor Adibah Wan Zakaria & normah Mohd noor carnivorous pitcher plants produce specialised pitcher organs containing secretory glands, which secrete acidic fluids with hydrolytic enzymes for prey digestion and nutrient absorption. The content of pitcher fluids has been the focus of many fluid protein profiling studies. These studies suggest an evolutionary convergence of a conserved group of similar enzymes in diverse families of pitcher plants. A recent study showed that endogenous proteins were replenished in the pitcher fluid, which indicates a feedback mechanism in protein secretion. This poses an interesting question on the physiological effect of plant protein loss. However, there is no study to date that describes the pitcher response to endogenous protein depletion. To address this gap of knowledge, we previously performed a comparative RNA-sequencing experiment of newly opened pitchers (D0) against pitchers after 3 days of opening (D3C) and pitchers with filtered endogenous proteins (>10 kDa) upon pitcher opening (D3L). Nepenthes ampullaria was chosen as a model study species due to their abundance and unique feeding behaviour on leaf litters. The analysis of unigenes with top 1% abundance found protein translation and stress response to be overrepresented in D0, compared to cell wall related, transport, and signalling for D3L. Differentially expressed gene (DEG) analysis identified DEGs with functional enrichment in protein regulation, secondary metabolism, intracellular trafficking, secretion, and vesicular transport. the transcriptomic landscape of the pitcher dramatically shifted towards intracellular transport and defence response at the expense of energy metabolism and photosynthesis upon endogenous protein depletion. This is supported by secretome, transportome, and transcription factor analysis with RT-qPCR validation based on independent samples. This study provides the first glimpse into the molecular responses of pitchers to protein loss with implications to future cost/benefit analysis of carnivorous pitcher plant energetics and resource allocation for adaptation in stochastic environments.Carnivorous plants are commonly found in habitats deprived of nutrients, especially nitrogen and phosphorus. To qualify as carnivorous, a plant must have at least one adaptation for active attraction, capture, digestion, and clear nutritional benefit from carnivory 1 . The convergent evolution of passive pitfall traps resulted in carnivorous pitcher plants of diverse orders and families, including Nepenthaceae (Caryophyllales), Cephalotaceae (Oxalidales), and Sarraceniaceae (Ericales) at distant geographical locations but sharing similar digestive fluid proteins 2-4 . A species-rich monotypic group of tropical pitcher plants from genus Nepenthes develop attractive pitchers of diverse shapes and sizes 5 . Recently, two highly conserved leaf developmental regulatory genes, ASYMMETRIC LEAVES1 (AS1) and REVOLUTA (REV), have been shown to be key in pitcher developme...

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

“…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 ). Proteins involved in secondary metabolism were also reported to be important for response against environmental stress such as pathogen attack that led to the synthesis of secondary metabolites from different pathways ( Chini et al, 2018 ; Goh 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 ). Proteins involved in secondary metabolism were also reported to be important for response against environmental stress such as pathogen attack that led to the synthesis of secondary metabolites from different pathways ( Chini et al, 2018 ; Goh et al, 2020 ). Further studies are needed to ascertain the roles of these proteins in secondary metabolism and stress response.…”

“…In comparison, knowledge of nutrient uptake and transportation is more limited. Furthermore, the regulatory mechanism of protein secretion and replenishment remains poorly understood ( Wan Zakaria et al, 2016b , 2019 ; Goh et al, 2020 ). Protein identification in the pitcher fluids is limited by the unusual amino acid composition and the limited sequence information for Nepenthes ( Lee et al, 2016 ).…”

Protease activity is maintained in Nepenthes ampullaria digestive fluids depleted of endogenous proteins with compositional changes

“…D0 and D3C samples were covered with drawstring gauze bags to prevent insect contamination but not for D3 and D7 samples. Some of the D0 pitchers were refilled with the pitcher fluid filtrates after protein extraction, covered with drawstring gauze bags, and collected again on day 3 as the D3L samples for endogenous protein depletion experiment (Goh et al 2020). An overview of the experimental workflow is depicted in Figure S1.…”

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

“…Studies of Nepenthes have largely focused on the proteins of pitcher fluids (Ravee et al, 2018). Recent studies have reported the effects of endogenous protein depletion on protein secretion in pitcher fluids of N. × ventrata (Wan Zakaria et al, 2019) and gene expression in pitchers of N. ampullaria with transcriptome analysis (Goh et al, 2020). The same proteomics informed by the transcriptomics approach was applied to explore the expression of genes and proteins in the pitchers and fluids, respectively, in N. ampullaria, N. rafflesiana, and their hybrid N. × hookeriana (Zulkapli et al, 2017;Zulkapli et al, 2021).…”