Abstract
Main conclusion
Nepenthes regulates enzyme activities by sensing stimuli from the insect prey. Protein is the best inductor mimicking the presence of an insect prey.
Carnivorous plants of the genus Nepenthes have evolved passive pitcher traps for prey capture. In this study, we investigated the ability of chemical signals from a prey (chitin, protein, and ammonium) to induce transcription and synthesis of digestive enzymes in Nepenthes × Mixta. We used real-time PCR and specific antibodies generated against the aspartic proteases nepenthesins, and type III and type IV chitinases to investigate the induction of digestive enzyme synthesis in response to different chemical stimuli from the prey. Transcription of nepenthesins was strongly induced by ammonium, protein and live prey; chitin induced transcription only very slightly. This is in accordance with the amount of released enzyme and proteolytic activity in the digestive fluid. Although transcription of type III chitinase was induced by all investigated stimuli, a significant accumulation of the enzyme in the digestive fluid was found mainly after protein and live prey addition. Protein and live prey were also the best inducers for accumulation of type IV chitinase in the digestive fluid. Although ammonium strongly induced transcription of all investigated genes probably through membrane depolarization, strong acidification of the digestive fluid affected stability and abundance of both chitinases in the digestive fluid. The study showed that the proteins are universal inductors of enzyme activities in carnivorous pitcher plants best mimicking the presence of insect prey. This is not surprising, because proteins are a much valuable source of nitrogen, superior to chitin. Extensive vesicular activity was observed in prey-activated glands.
Similar content being viewed by others
Abbreviations
- AP:
-
Aspartic protease
References
Adlassnig W, Koller-Peroutka M, Bauer S, Koshkin E, Lendl T, Lichtscheidl IK (2012) Endocytotic uptake of nutrients in carnivorous plants. Plant J 71:303–313
An CI, Fukusaki EI, Kobayashi A (2001) Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco. Planta 212:547–555
Athauda SBP, Matsumoto K, Rajapakshe S, Kuribayashi M, Kojima M, Kubomura-Yoshida N, Iwamatsu A, Shibata C, Inoue H, Takahashi K (2004) Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases. Biochem J 381:295–306
Baby S, Johnson AJ, Zachariah EJ, Hussain AA (2017) Nepenthes pitchers are CO2-enriched cavities, emit CO2 to attract preys. Sci Rep 7:11281
Bazile V, Moran JA, Le Moguédec G, Marshall DJ, Gaume L (2012) A carnivorous plant fed by its ant symbiont: a unique multi-faceted nutritional mutualism. PLoS ONE 7:e36179
Bemm F, Becker D, Larisch C, Kreuzer I, Escalante-Perez M, Schulze WX, Ankenbrand M, Van De Weyer AL, Krol E, Al-Rasheid KA, Mithöfer A, Weber AP, Schultz J, Hedrich R (2016) Venus flytrap carnivorous lifestyle builds on herbivore defense strategies. Genome Res 26:812–825
Biteau F, Nisse E, Miguel S, Hannewald P, Bazile V, Gaume L, Mignard B, Hehn A, Bourgaud F (2013) A simple SDS-PAGE protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae). Am J Bot 100:2478–2484
Böhm J, Scherzer S, Krol E, Kreuzer I, Meyer K, Lorey C, Mueller TD, Shabala L, Monte I, Solano R, Al-Rasheid KAS, Rennenberg H, Shabala S, Neher E, Hedrich R (2016) The Venus flytrap Dionaea muscipula counts prey-induced action potentials to induce sodium uptake. Curr Biol 26:286–295
Bonhomme V, Gounand I, Alaux C, Jousselin E, Barthélémy D, Gaume L (2011) The plant-ant Camponotus schmitzi helps its carnivorous host-plant Nepenthes bicalcarata to catch its prey. Trop Ecol 27:15–24
Buch F, Rott M, Rottloff S, Paetz C, Hilke I, Raessler M, Mithöfer A (2013) Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth. Ann Bot 111:375–383
Buch F, Kaman WE, Bikker FJ, Yilamujiang A, Mithöfer A (2015) Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous Nepenthes plants. PLoS ONE 10:e0118853
Clarke C (1997) Nepenthes of borneo. Natural History Publication, Kota Kinabalu
Clarke CM, Bauer U, Lee CC, Tuen AA, Rembold K, Moran JA (2009) Tree shrew lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant. Biol Lett 5:632–635
Drakakaki G, Dandekar A (2013) Show more protein secretion: how many secretory routes does a plant cell have? Plant Sci 203–204:74–78
Eilenberg H, Pnini-Cohen S, Schuster S, Movtchan A, Zilberstein A (2006) Isolation and characterization of chitinase genes from pitchers of the carnivorous plant Nepenthes khasiana. J Exp Bot 57:2775–2784
Eilenberg H, Pnini-Cohen S, Rahamim Y, Sionov E, Segal E, Carmeli S, Zilberstein A (2010) Induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant Nepenthes khasiana. J Exp Bot 61:911–922
Frazier CK (2000) The enduring controversies concerning the process of protein digestion in Nepenthes (Nepenthaceae). Carnivorous Plant Newsletter 29:56–61
Fukushima K, Fang X, Alvarez-Ponce D, Cai H, Carretero-Paulet L, Chen C, Chang T-H, Farr KM, Fujita T, Hiwatashi Y et al (2017) Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory. Nat Ecol Evol 1:0059
Gallie DR, Chang S-C (1997) Signal transduction in the carnivorous plant Sarracenia purpurea. Plant Physiol 115:1461–1471
Grafe TU, Schöner CR, Kerth G, Junaidi A, Schöner MG (2011) A novel resource–service mutualism between bats and pitcher plants. Biol Lett 7:436–439
Hatano N, Hamada T (2008) Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata. J Proteome Res 7:809–816
Hatano N, Hamada T (2012) Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata. J Proteomics 75:4844–4852
Higashi S, Nakashima A, Ozaki H, Abe M (1993) Analysis of feeding mechanism in pitcher of Nepenthes hybrida. J Plant Res 106:47–54
Hooker JD (1874) The carnivorous habits of plants. Nature 10:366–372
Ilík P, Hlaváčková V, Krchňák P, Nauš J (2010) A low-noise multi-channel device for the monitoring of systemic electrical signal propagation in plants. Biol Plant 54:185–190
Ishisaki K, Arai S, Hamada T, Honda Y (2012a) Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata. Carbohydr Res 361:170–174
Ishisaki K, Honda Y, Taniguchi H, Hatano N, Hamada T (2012b) Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata. Glycobiology 22:345–351
Jopcik M, Moravcikova J, Matusikova I, Bauer M, Rajninec M, Libantova J (2017) Structural and functional characterisation of a class I endochitinase of the carnivorous sundew (Drosera rotundifolia L.). Planta 245:313–327
Juniper BE, Robins RJ, Joel DM (1989) The carnivorous plants. Academic Press, London
Kadek A, Tretyachenko V, Mrazek H, Ivanova I, Halada P, Rey M, Schriemer DC, Man P (2014) Expression and characterization of plant aspartic proteases nepenthesin-1 from Nepenthes gracilis. Protein Expres Purif 95:121–128
Krausko M, Perutka Z, Šebela M, Šamajová O, Šamaj J, Novák O, Pavlovič A (2017) The role of electrical and jasmonate signalling in the recognition of captured prey in the carnivorous sundew plant Drosera capensis. New Phytol 213:1818–1835
Lee L, Zhang Y, Ozar B, Sensen CW, Schriemer DC (2016) Carnivorous nutrition in pitcher plants (Nepenthes spp.) via an unusual complement of endogenous enzymes. J Proteome Res 15:3108–3117
Libiaková M, Floková K, Novák O, Slováková L, Pavlovič A (2014) Abundance of cysteine endopeptidase dionain in digestive fluid of Venus flytrap (Dionaea muscipula Ellis) is regulated by different stimuli from prey through jasmonates. PLoS ONE 9:e104424
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408
Maffei ME, Mithöfer A, Boland W (2007) Before gene expression: early events in plant-insect interaction. Trends Plant Sci 12:310–316
Matušíková I, Salaj J, Moravčíková J, Mlynárová L, Nap JP, Libantová J (2005) Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show induction of chitinase activity upon mimicking the presence of prey. Planta 222:1020–1027
Merbach MA, Merbach DJ, Maschwitz U, Booth WE, Fiala B, Zizka G (2002) Carnivorous plants: mass march of termites into the deadly trap. Nature 415:36–37
Merbach MA, Zizka G, Fiala B, Merbach DJ, Booth WE, Maschwitz U (2007) Why a carnivorous plant cooperates with an ant—selective defense against pitcher-destroying weevils in the myrmecophytic pitcher plant Nepenthes bicalcarata Hook.f. Ecotropica 13:45–56
Mithöfer A (2011) Carnivorous pitcher plants: insights in an old topic. Phytochemistry 72:1678–1682
Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA 104:19613–19618
Moran JA, Clarke CM, Hawkins BJ (2003) From carnivore to detritivore? Isotopic evidence for leaf litter utilization by the tropical pitcher plant Nepenthes ampullaria. Int J Plant Sci 164:635–639
Moran JA, Hawkins BJ, Gowen BE, Robbins SL (2010) Ion fluxes across the pitcher walls of three Bornean Nepenthes pitcher plant species: flux rates and gland distribution patterns reflect nitrogen sequestration strategies. J Exp Bot 61:1365–1374
Newmann M-A, Sundelin T, Nielsen JT, Erbs G (2013) MAMP (microbe-associated molecular pattern) triggered immunity in plants. Front Plant Sci 4:139
Pavlovič A, Slováková Ľ, Šantrůček J (2011) Nutritional benefit from leaf litter utilization in the pitcher plants Nepenthes ampullaria. Plant Cell Environ 34:1865–1873
Pavlovič A, Krausko M, Adamec L (2016) A carnivorous sundew plant prefers protein over chitin as a source of nitrogen from its traps. Plant Physiol Biochem 104:11–16
Pavlovič A, Jakšová J, Novák O (2017) Triggering a false alarm: wounding mimics prey capture in the carnivorous Venus flytrap (Dionaea muscipula). New Phytol 216:927–938
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:2003–2007
Rawat S, Ali S, Mittra B, Grover A (2017) Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea. Biotechnol Rep 13:72–79
Renner T, Specht CD (2012) Molecular and functional evolution of class I chitinases for plant carnivory in the Caryophyllales. Mol Biol Evol 29:2971–2985
Rey M, Yang M, Lee L, Zhang Y, Sheff JG, Sensen CW, Mrazek H, Halada P, Man P, McCarville JL, Verdu EF, Schriemer DC (2016) Addresing proteolytic efficiency in enzymatic degradation therapy for celiac disease. Sci Rep 6:30980
Rottloff S, Stieber R, Maischak H, Turini FG, Heubl G, Mithöfer A (2011) Functional characterization of a class III acid chitinase from the traps of the carnivorous pitcher plant genus Nepenthes. J Exp Bot 62:4639–4647
Rottloff S, Miguel S, Biteau F, Nisse E, Hammann P, Kuhn L, Chicher J, Bazile V, Gaume L, Mignard B, Hehn A, Bourqaud F (2016) Proteome analysis of digestive fluids in Nepenthes pitchers. Ann Bot 117:479–495
Scharmann M, Thornham DG, Grafe TU, Federle W (2013) A novel type of nutritional ant–plant interaction: ant partners of carnivorous pitcher plants prevent nutrient export by dipteran pitcher infauna. PLoS ONE 8:e63556
Scherzer S, Krol E, Kreuzer I, Kruse J, Karl F, Von Rüden M, Escalante-Perez M, Müller T, Rennenberg H, Al-Rasheid KAS, Neher E, Hedrich R (2013) The Dionaea muscipula ammonium channel DmAMT1 provides NH4 + uptake associated with Venus flytrap’s prey digestion. Curr Biol 23:1649–1657
Scherzer S, Shabala L, Hedrich B, Fromm J, Bauer H, Munz E, Jakob P, Al-Rascheid KAS, Kreuzer I, Becker D, Eilbmeier M, Rennenberg H, Shabala S, Bennett M, Neher E, Hedrich R (2017) Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells. Proc Natl Acad Sci USA 114:4822–4827
Schulze W, Frommer WB, Ward JM (1999) Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes. Plant J 17:637–646
Takeuchi Y, Salcher MM, Ushio M, Shimizu-Inatsugi R, Kobayashi MJ, Diway B, von Mering C, Pernthaler J, Shimizu KK (2011) In situ enzyme activity in the dissolved and particulate fraction of the fluid from four pitcher plant species of the genus Nepenthes. PLoS ONE 6:e25144
Thornham DG, Smith JM, Grafe TU, Federle W (2012) Setting the trap: cleaning behaviour of Camponotus schmitzi ants increases long-term capture efficiency of their pitcher plant host, Nepenthes bicalcarata. Funct Ecol 26:11–19
Thorogood CJ, Bauer U, Hiscock SJ (2018) Convergent and divergent evolution in carnivorous pitcher plant traps. New Phytol 217:1035–1041
Williams SE, Pickard BG (1972) Properties of action potentials in Drosera tentacles. Planta 103:222–240
Yilamujiang A, Reichelt M, Mithöfer A (2016) Slow food: insect prey and chitin induce phytohormone accumulation and gene expression in carnivorous Nepenthes plants. Ann Bot 118:369–735
Yilamujiang A, Zhu A, Ligabue-Braun R, Bartram S, Witte CP, Hedrich R, Hasebe M, Schöner CR, Schöner MG, Kerth G, Carlini CR, Mithöfer A (2017) Coprophagous features in carnivorous Nepenthes plants: a task for ureases. Sci Rep 7:11647
Acknowledgements
This work was supported by the Czech Science Foundation Agency [project GAČR 16-07366Y] and the Ministry of Education, Youth and Sports of the Czech Republic through the National Program of Sustainability I [grant LO1204]. This publication is also the result of the project implementation: Comenius University in Bratislava Science Park supported by the Research and Development Operational Programme funded by the ERDF (Grant number: ITMS 26240220086). We thank Lukáš Nosek for the help with transmission electron microscope and two anonymous reviewers for their suggestions and comments.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
Inhibition of proteolytic activity by 15 μM pepstatin in Nepenthes × Mixta. Without inhibitor (closed cirles) and inhibitor added (open circles). Means ± SD, n = 3. Supplementary material 1 (TIFF 39 kb)
Fig. S2
Abundance of nepenthesins in the digestive fluid of Nepenthes x Mixta using specific antibody by Western blots before and 3, 6, and 9 days after feeding in the case where the initial level of nepenthesins in the digestive fluid was high. The same volume of digestive fluid was electrophoresed. Supplementary material 2 (TIFF 72 kb)
Fig. S3
Autoactivation of nepenthesins at acidic pH in Nepenthes truncata. The digestive fluid was collected from freshly opened pitcher, transferred into the Falcon tube and its pH was measured (pH 6.7). Then, the volume was divided and half of the fluid was acidified to pH 2 by adding HCl. Both samples were incubated in Falcon tubes for 24 h at room temperature and then the same volume of the fluid was electrophoresed and blotted on nitrocellulose membrane. Antibody against nepenthesin was used. No shift in electrophoretic mobility was detected, indicating that nepenthesin was already autoactivated in control pitchers. Supplementary material 3 (TIFF 32 kb)
Fig. S4
Stability of enzymes at acidic conditions in Nepenthes x Mixta. The empty pitchers were fed and the digestive fluid was collected on the third day and divided into two samples. The same volume of 200 mM glycine-HCl buffer at pH 1.5 and 4 was added and the samples were incubated at room temperature for 24 and 72 h. The same volume was electrophoresed and blotted on nitrocellulose membrane and incubated with antibodies against nepenthesin and type III and IV chitinases. The abundance of chitinases was decreased at acidic conditions in comparisons to nepenthesins. Lane C represents a control pitcher before prey addition. Supplementary material 4 (TIFF 337 kb)
Rights and permissions
About this article
Cite this article
Saganová, M., Bokor, B., Stolárik, T. et al. Regulation of enzyme activities in carnivorous pitcher plants of the genus Nepenthes. Planta 248, 451–464 (2018). https://doi.org/10.1007/s00425-018-2917-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-018-2917-7