Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants

Abstract

Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

Venus flytrap carnivorous lifestyle builds on herbivore defense strategies

“…Activation of the JA pathway leads to the processing and consumption of, from the view of a noncarnivorous plant, a potential herbivore. Prey capture of the sundew Drosera induces both leaf bending and the accumulation of defense-related jasmonate phytohormones (Nakamura et al 2013), and jasmonate increases the proteolytic activity in the pitcher fluid of the Nepenthes plant (Buch et al 2015). Taken together, this suggests that Dionaea rewires defense responses known from noncarnivorous plants in order to operate its carnivorous lifestyle and that jasmonate signaling probably represents a basic mechanism in plant carnivory (Fig.…”

“…This is also the case for insect-stimulated traps of the carnivorous plant Drosera carpensis (Nakamura et al 2013). JA not only represents a chemical trigger of gland cell secretion (Libiakova et al 2014;Buch et al 2015) but at the transcriptional level is able to partially substitute for insect action (see Figs. 3, 4A,B).…”

“…Initially, Dionaea invests energy in producing and growing traps and in providing the morphological and anatomical requirements for fast insect capture by storing elastic energy in a metastable configuration of the snap trap (Forterre et al 2005). During this nonstimulated phase, glands are already producing hydrolases such as the Nepenthesin-like aspartic protease (Buch et al 2015). Upon insect capture, this pH-and temperature-resistant broad range protease is probably among the first hydrolases released, with others being massively induced after chemical sensing and ongoing prey digestion.…”

“…3A, denominators; Supplemental Table S7). Among these, we found transcripts encoding the Nepenthesin-like aspartic protease (Buch et al 2015) and a Lipid Transfer Protein (LTP). Both these genes were already highly expressed in nonstimulated glands, but the majority of hydrolase genes showed a massive induction ranging from fivefold to 900-fold following stimulation (Fig.…”

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity

“…[5]. The fluid is composed of a mixture of enzymes generated by the plants themselves including aspartic proteases called nepenthesins [6,7] and those produced from inhabitant microorganisms [8]. In addition to proteases, the pitcher fluids of Nepenthes also contain esterases, phosphatases, ribonucleases, and chitinases [9][10][11][12][13] as well as different glycosyl hydrolases, e.g., β-D-glucosidases and β-D-glucosaminidases [8] which together act cooperatively on digesting the insect bodies.…”