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.

Department of Biophysics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Šlechtitelů 27 783 71 Olomouc Czech Republic

Department of Plant Physiology Faculty of Natural Sciences Comenius University in Bratislava Ilkovičova 6 Mlynská dolina B2 842 15 Bratislava Slovakia

Department of Plant Physiology Faculty of Natural Sciences Comenius University in Bratislava Ilkovičova 6 Mlynská dolina B2 842 15 Bratislava Slovakia Comenius University Science Park Comenius University in Bratislava Ilkovičova 8 841 04 Bratislava Slovakia

Department of Plant Physiology Plant Science and Biodiversity Centre Slovak Academy of Sciences Dúbravská cesta 9 845 23 Bratislava Slovakia Department of Biophysics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Šlechtitelů 27 783 71 Olomouc Czech Republic

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