A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid of Dionaea muscipula (Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus’s flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS–PAGE band protein. The name “dionain” is proposed for the enzyme.     

Abundance of Cysteine Endopeptidase Dionain in Digestive Fluid of Venus Flytrap (Dionaea muscipula Ellis) Is Regulated by Different Stimuli from Prey through Jasmonates

The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey''s movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH₄Cl, KH₂PO₄, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed.     

Energetics and forces of the Dionaea muscipula trap closing

The Venus flytrap is the most famous carnivorous plant. The electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf in 0.3 s without mechanical stimulation of trigger hairs. Here we present results for direct measurements of the closing force of the trap of Dionaea muscipula Ellis after mechanical or electrical stimulation of the trap using the piezoelectric thin film or Fuji Prescale indicating sensor film. The closing force was 0.14 N and the corresponding pressure between rims of two lobes was 38 kPa. We evaluated theoretically using the Hydroelastic Curvature Model and compared with experimental data velocity, acceleration and kinetic energy from the time dependencies of distance between rims of lobes during the trap closing. The Charge Stimulation Method was used for trap electrostimulation between the midrib and lobes. From the dependence of voltage between two Ag/AgCl electrodes in the midrib and one of the lobes, we estimated electrical charge, current, resistance, electrical energy and electrical power dependencies on time during electrostimulation of the trap.     

A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap)

The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme.     

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S₂ and for positively charged residues in S₁. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S₂ pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.     

Trap diversity and evolution in the family Droseraceae

We review trapping mechanisms in the carnivorous flowering plant family Droseraceae (order Caryophyllales). Its members are generally known to attract, capture, retain and digest prey animals (mainly arthropods) with active snap-traps (Aldrovanda, Dionaea) or with active sticky flypaper traps (Drosera) and to absorb the resulting nutrients. Recent investigations revealed how the snap-traps of Aldrovanda vesiculosa (waterwheel plant) and Dionaea muscipula (Venus’ flytrap) work mechanically and how these apparently similar devices differ as to their functional morphology and shutting mechanics. The Sundews (Drosera spp.) are generally known to possess leaves covered with glue-tentacles that both can bend toward and around stuck prey. Recently, it was shown that there exists in this genus a higher diversity of different tentacle types and trap configurations than previously known which presumably reflect adaptations to different prey spectra. Based on these recent findings, we finally comment on possible ways for intrafamiliar trap evolution.     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

Complete hunting cycle of Dionaea muscipula: Consecutive steps and their electrical properties

The total hunting cycle of the Venus flytrap consists of five stages: 1. Open state → 2. Closed state → 3. Locked state → 4. Constriction and digestion → 5. Semi-open state → 1. Open state. The opening of the trap after digestion consists of two steps: opening of the lobes, and changing of their curvature from concave to convex shape. Uncouplers carbonylcyanide-4-trifluoromethoxyphenyl hydrazone (FCCP) and carbonylcyanide-3-chlorophenylhydrazone (CCCP) inhibit the trap from opening for two weeks and antracene-9-carboxylic acid inhibits the trap from constricting. Different stages of the hunting cycle have different electrical characteristics. The biologically closed electrochemical circuits in the Venus flytrap are analyzed using the charged capacitor method. If the initial voltage applied to the Venus flytrap is 0.5 V or greater, changing the polarity of the electrodes between the midrib and one of the lobes results in a rectification effect and in different kinetics of discharge capacitance. These effects can be caused by the fast transport of ions through ion channels. The electrical properties of the Venus flytrap were investigated and equivalent electrical circuits within the upper leaf were proposed to explain the experimental data.     

Electrotonic and action potentials in the Venus flytrap

The electrical phenomena and morphing structures in the Venus flytrap have attracted researchers since the nineteenth century. We have observed that mechanical stimulation of trigger hairs on the lobes of the Venus flytrap induces electrotonic potentials in the lower leaf. Electrostimulation of electrical circuits in the Venus flytrap can induce electrotonic potentials propagating along the upper and lower leaves. The instantaneous increase or decrease in voltage of stimulating potential generates a nonlinear electrical response in plant tissues. Any electrostimulation that is not instantaneous, such as sinusoidal or triangular functions, results in linear responses in the form of small electrotonic potentials. The amplitude and sign of electrotonic potentials depend on the polarity and the amplitude of the applied voltage. Electrical stimulation of the lower leaf induces electrical signals, which resemble action potentials, in the trap between the lobes and the midrib. The trap closes if the stimulating voltage is above the threshold level of 4.4 V. Electrical responses in the Venus flytrap were analyzed and reproduced in the discrete electrical circuit. The information gained from this study can be used to elucidate the coupling of intracellular and intercellular communications in the form of electrical signals within plants.     

Closing of Venus Flytrap by Electrical Stimulation of Motor Cells

Electrical signaling and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of researchers since XIX century, but the exact mechanism of Venus flytrap closure is still unknown. We found that the electrical stimulus between a midrib and a lobe closes the Venus flytrap leaf by activating motor cells without mechanical stimulation of trigger hairs. The closing time of Venus flytrap by electrical stimulation of motor cells is 0.3 s, the same as mechanically induced closing. The mean electrical charge required for the closure of the Venus flytrap leaf is 13.6 µC. Ion channel blockers such as Ba²⁺, TEACl as well as uncouplers such as FCCP, 2,4-dinitrophenol and pentachlorophenol dramatically decrease the speed of the trap closing. Using an ultra-fast data acquisition system with measurements in real time, we found that the action potential in the Venus flytrap has a duration time of about 1.5 ms. Our results demonstrate that electrical stimulation can be used to study mechanisms of fast activity in motor cells of the plant kingdom.Key Words: action potential, electrophysiology, electrical signaling, Venus flytrap, motor cells     

Traps of carnivorous pitcher plants as a habitat: composition of the fluid, biodiversity and mutualistic activities

BACKGROUND: Carnivorous pitcher plants (CPPs) use cone-shaped leaves to trap animals for nutrient supply but are not able to kill all intruders of their traps. Numerous species, ranging from bacteria to vertrebrates, survive and propagate in the otherwise deadly traps. This paper reviews the literature on phytotelmata of CPPs. PITCHER: Fluid as a Habitat The volumes of pitchers range from 0·2 mL to 1·5 L. In Nepenthes and Cephalotus, the fluid is secreted by the trap; the other genera collect rain water. The fluid is usually acidic, rich in O(2) and contains digestive enzymes. In some taxa, toxins or detergents are found, or the fluid is extremely viscous. In Heliamphora or Sarracenia, the fluid differs little from pure water. INQUILINE: Diversity Pitcher inquilines comprise bacteria, protozoa, algae, fungi, rotifers, crustaceans, arachnids, insects and amphibia. The dominant groups are protists and Dipteran larvae. The various species of CPPs host different sets of inquilines. Sarracenia purpurea hosts up to 165 species of inquilines, followed by Nepenthes ampullaria with 59 species, compared with only three species from Brocchinia reducta. Reasons for these differences include size, the life span of the pitcher as well as its fluid. MUTUALISTIC: Activities Inquilines closely interact with their host. Some live as parasites, but the vast majority are mutualists. Beneficial activities include secretion of enzymes, feeding on the plant's prey and successive excretion of inorganic nutrients, mechanical break up of the prey, removal of excessive prey and assimilation of atmospheric N(2). CONCLUSIONS: There is strong evidence that CPPs influence their phytotelm. Two strategies can be distinguished: (1) Nepenthes and Cephalotus produce acidic, toxic or digestive fluids and host a limited diversity of inquilines. (2) Genera without efficient enzymes such as Sarracenia or Heliamphora host diverse organisms and depend to a large extent on their symbionts for prey utilization.     

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant,Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.     

Trap closure and prey retention in Venus flytrap (Dionaea muscipula) temporarily reduces photosynthesis and stimulates respiration

Background and Aims

The carnivorous plant Venus flytrap (Dionaea muscipula) produces a rosette of leaves: each leaf is divided into a lower part called the lamina and an upper part, the trap, with sensory trigger hairs on the adaxial surface. The trap catches prey by very rapid closure, within a fraction of a second of the trigger hairs being touched twice. Generation of action potentials plays an important role in closure. Because electrical signals are involved in reduction of the photosynthetic rate in different plant species, we hypothesized that trap closure and subsequent movement of prey in the trap will result in transient downregulation of photosynthesis, thus representing the energetic costs of carnivory associated with an active trapping mechanism, which has not been previously described.

Methods

Traps were enclosed in a gas exchange cuvette and the trigger hairs irritated with thin wire, thus simulating insect capture and retention. Respiration rate was measured in darkness (R_D). In the light, net photosynthetic rate (A_N), stomatal conductance (g_s) and intercellular CO₂ concentration (c_i) were measured, combined with chlorophyll fluorescence imaging. Responses were monitored in the lamina and trap separately.

Key Results

Irritation of trigger hairs resulted in decreased A_N and increased R_D, not only immediately after trap closure but also during the subsequent period when prey retention was simulated in the closed trap. Stomatal conductance remained stable, indicating no stomatal limitation of A_N, so c_i increased. At the same time, the effective quantum yield of photosystem II (Φ_PSII) decreased transiently. The response was confined mainly to the digestive zone of the trap and was not observed in the lamina. Stopping mechanical irritation resulted in recovery of A_N, R_D and Φ_PSII.

Conclusions

We put forward the first experimental evidence for energetic demands and carbon costs during insect trapping and retention in carnivorous plants, providing a new insight into the cost/benefit model of carnivory.     

Biologically Closed Electrical Circuits in Venus Flytrap

The Venus flytrap (Dionaea muscipula Ellis) is a marvel of plant electrical, mechanical, and biochemical engineering. The rapid closure of the Venus flytrap upper leaf in about 0.1 s is one of the fastest movements in the plant kingdom. We found earlier that the electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf without mechanical stimulation of trigger hairs. The Venus flytrap can accumulate small subthreshold charges and, when the threshold value is reached, the trap closes. Here, we investigated the electrical properties of the upper leaf of the Venus flytrap and proposed the equivalent electrical circuit in agreement with the experimental data.     

Jasmonates trigger prey-induced formation of ‘outer stomach’ in carnivorous sundew plants

It has been widely accepted that the growth-related phytohormone auxin is the endogenous signal that initiates bending movements of plant organs. In 1875, Charles Darwin described how the bending movement of leaves in carnivorous sundew species formed an ‘outer stomach’ that allowed the plants to enclose and digest captured insect prey. About 100 years later, auxin was suggested to be the factor responsible for this movement. We report that prey capture induces both leaf bending and the accumulation of defence-related jasmonate phytohormones. In Drosera capensis fed with fruitflies, within 3 h after prey capture and simultaneous with leaf movement, we detected an increase in jasmonic acid and its isoleucine conjugate. This accumulation was spatially restricted to the bending segment of the leaves. The application of jasmonates alone was sufficient to trigger leaf bending. Only living fruitflies or the body fluids of crushed fruitflies induced leaf curvature; neither dead flies nor mechanical treatment had any effect. Our findings strongly suggest that the formation of the ‘outer stomach’ in Drosera is a chemonastic movement that is triggered by accumulation of endogenous jasmonates. These results suggest that in carnivorous sundew plants the jasmonate cascade might have been adapted to facilitate carnivory rather than to defend against herbivores.     

Capture of algae promotes growth and propagation in aquatic Utricularia

Background and Aims Some carnivorous plants trap not only small animals but also algae and pollen grains. However, it remains unclear if these trapped particles are useless bycatch or whether they provide nutrients for the plant. The present study examines this question in Utricularia, which forms the largest and most widely spread genus of carnivorous plants, and which captures prey by means of sophisticated suction traps.Methods Utricularia plants of three different species (U. australis, U. vulgaris and U. minor) were collected in eight different water bodies including peat bogs, lakes and artificial ponds in three regions of Austria. The prey spectrum of each population was analysed qualitatively and quantitatively, and correlated with data on growth and propagation, C/N ratio and δ¹⁵N.Key Results More than 50 % of the prey of the Utricularia populations investigated consisted of algae and pollen, and U. vulgaris in particular was found to capture large amounts of gymnosperm pollen. The capture of algae and pollen grains was strongly correlated with most growth parameters, including weight, length, budding and elongation of internodes. The C/N ratio, however, was less well correlated. Other prey, such as moss leaflets, fungal hyphae and mineral particles, were negatively correlated with most growth parameters. δ¹⁵N was positively correlated with prey capture, but in situations where algae were the main prey objects it was found that the standard formula for calculation of prey-derived N was no longer applicable.Conclusions The mass capture of immotile particles confirms the ecological importance of autonomous firing of the traps. Although the C/N ratio was little influenced by algae, they clearly provide other nutrients, possibly including phosphorus and trace elements. By contrast, mosses, fungi and mineral particles appear to be useless bycatch. Correlations with chemical parameters indicate that Utricularia benefits from nutrient-rich waters by uptake of inorganic nutrients from the water, by the production of more traps per unit of shoot length, and by the capture of more prey particles per trap, as nutrient-rich waters harbour more prey organisms.     

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.     

Red trap colour of the carnivorous plant Drosera rotundifolia does not serve a prey attraction or camouflage function

The traps of many carnivorous plants are red in colour. This has been widely hypothesized to serve a prey attraction function; colour has also been hypothesized to function as camouflage, preventing prey avoidance. We tested these two hypotheses in situ for the carnivorous plant Drosera rotundifolia. We conducted three separate studies: (i) prey attraction to artificial traps to isolate the influence of colour; (ii) prey attraction to artificial traps on artificial backgrounds to control the degree of contrast and (iii) observation of prey capture by D. rotundifolia to determine the effects of colour on prey capture. Prey were not attracted to green traps and were deterred from red traps. There was no evidence that camouflaged traps caught more prey. For D. rotundifolia, there was a relationship between trap colour and prey capture. However, trap colour may be confounded with other leaf traits. Thus, we conclude that for D. rotundifolia, red trap colour does not serve a prey attraction or camouflage function.