Regulation of enzyme activities in carnivorous pitcher plants of the genus Nepenthes

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.

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Abbreviations

AP:

Aspartic protease

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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.

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Correspondence to Andrej Pavlovič.

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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)

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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

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Keywords

  • Carnivorous plant
  • Chitin
  • Chitinase
  • Enzyme
  • Nepenthesin
  • Pitcher plant
  • Protease