The content of pitcher fluids has been the focus of many fluid protein profiling studies, which suggest an evolutionary convergence of a conserved group of similar enzymes in diverse families of pitcher plants.
Proteomics profiling of pitcher fluids in N. x ventrata 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 transcriptomic response to endogenous protein depletion. To address this gap of knowledge, we 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).
Proteomics profiling of pitcher fluids in N. x ventrata 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 transcriptomic response to endogenous protein depletion. To address this gap of knowledge, we 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).
The aim of this study is to generate functional annotations for the first reference transcriptome of N. ampullaria pitchers for further molecular studies.
By using N. ampullaria as a model system to study carnivorous plant protein depletion, we address the following questions:
|
Nepenthes ampullaria was chosen as a model study species due to their abundance and unique feeding behaviour (detritivory) on leaf litters.
Transcriptomics Analysis
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.
Significance
One strength of this protein depletion experiment was the use of filtrate to minimise the confounding effects from the loss of ions or metabolites in the pitcher fluids and to prevent pitcher senescence. A great challenge was the on-site monitoring of pitcher development to capture the timing of pitcher opening for performing this experiment in a controlled manner. The same experiment was independently repeated for RT-qPCR validation of gene expression. Therefore, the results obtained from the RNA-seq analysis are reliable and provide valuable data for subsequent in-depth experiments. This study as summarised in Fig. S8 contributes significantly to our knowledge and understanding of molecular responses of carnivory organs during protein loss in pitcher plants. Overall, our study suggests a trade-off between photosynthesis, resource allocation, and organ defence during endogenous protein replenishment in the pitcher fluids.
Proteomics Analysis
We tracked the pitcher development and pooled six biological replicates to profile endogenous proteins from newly opened pitchers within 24 h of lid opening (D0) and after 3 days of opening (D3C) through gel‐free liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomics. Based on N. ampullaria pitcher transcriptomes from previous Illumina (Wan Zakaria et al. 2016b) and PacBio (Zulkapli et al. 2017) sequencing, we identified the pitcher fluid proteins (secretome). Furthermore, we also conducted a controlled protein depletion experiment (D3L) to study the effects of endogenous protein depletion on pitcher fluid protein composition and protease activity by including uncovered day 3 (D3) and day 7 (D7) samples.
We provided comprehensive profiling of N. ampullaria pitcher fluid proteins to investigate the pitcher response to endogenous protein depletion. The protein sequence database generated from the previous transcriptome studies facilitated protein identification from the pitcher fluids via LC–MS/MS analysis for functional inference of protein compositional changes. The protease activity assays supported the replenishment of proteases in the pitcher fluids to a level comparable to that of control samples. This suggests a feedback mechanism with signaling in the pitcher tissue, which regulates the secretion of enzymes to maintain hydrolytic activities upon protein loss in the pitcher fluids. The regulatory mechanism of protein turnover inside the pitcher fluids warrants further studies.
Unlike most investigations on the feeding effects of different nitrogenous substrates on the induction of enzyme secretion, this study and a recent report on N. × ventrata (Wan Zakaria et al. 2016a; Wan Zakaria et al. 2019) are uncommon to experiment on the dynamics of endogenous secretome that is unique to botanical carnivory. Pitfall traps of carnivorous plants provide a unique model for the cost–benefit investigation of secretome homeostasis and studying the regulation of plant protein secretion that is analogous to the animal digestive system.
Significance
We demonstrated the replenishment of certain hydrolytic enzymes and the recovery of protease activity in the pitcher fluids within three days during young pitcher maturation. Such observations raise many intriguing questions: Are the proteins still replenished in the mature pitchers? How do the pitcher plants measure or detect the content of proteases in the secreted fluids? Do different molecular components regulate the secretion of different enzymes? How do the plants gauge and maintain proteostasis and different enzyme activities? Solving these mysteries will contribute towards the biotechnological development of pitchers as bioreactors for plant‐made proteins from Darwin's most wonderful plants.
References
Grants
- Ravee R, Baharin A, Cho W-T, Ting T-Y & Goh H-H* (2021) Protease activity is maintained in Nepenthes ampullaria digestive fluids depleted of endogenous proteins with compositional changes. Physiologia Plantarum doi: 10.1111/ppl.13540
- Goh H-H*, Baharin A, Mohd Salleh FI, Ravee R, Wan Zakaria WNA. & Mohd Noor N (2020) Transcriptome-wide shift from photosynthesis and energy metabolism upon endogenous fluid protein depletion in young Nepenthes ampullaria pitchers. Scientific Reports 10: 6575.
- Zulkapli MM, Rosli AF, Mohd Salleh F-‘I, Normah MN, Aizat WM & Goh H-H* (2016) Iso-Seq analysis of Nepenthes ampullaria, Nepenthes rafflesiana and Nepenthes x hookeriana for hybridisation study in pitcher plants. Genomics Data, 12, 130-131.
- Wan Zakaria WA, Loke K-K, Zulkapli MM, Mohd Salleh F-‘I, Goh H-H* & Normah M.N. (2016) RNA-seq analysis of Nepenthes ampullaria. Frontiers in Plant Science, 6, 1229.
- Wan Zakaria WNA., Aizat WM, Goh H-H* & Mohd Noor N (2019) Protein replenishment in pitcher fluids of Nepenthes × ventrata revealed by quantitative proteomics (SWATH-MS) informed by transcriptomics. Journal of Plant Research 132 (5), 681-694.
Grants
- FUNCTIONAL ANALYSIS OF A NOVEL PROLYL PROTEASE NEPROSIN FROM Nepenthes ampullaria - FRGS/1/2019/STG05/UKM/02/10 [1 Sep 2019 – 31 Aug 2021]
- GENOME-WIDE IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF NOVEL PROTEINS FROM Nepenthes ampullaria THROUGH TRANSCRIPTOMIC APPROACH - FRGS/2/2014/SG05/UKM/02/4 [1 Dec 2014 – 30 Nov 2017]
Copyright © 2022 UKM