As prey and pollinators, insects increase reproduction and allow for outcrossing in the carnivorous plant Dionaea muscipula.

PREMISE: Understanding the factors that limit reproductive success is a key component of plant biology. Carnivorous plants rely on insects as both nutrient sources and pollinators, providing a unique system for studying the effects of both resource and pollen limitation on plant reproduction. METHODS: We conducted a field experiment using wild-growing Dionaea muscipula J. Ellis (Droseraceae) in which we manipulated prey and pollen in a factorial design and measured flower production, number of fruits, and number of seeds. Because understanding reproduction requires knowledge of a plant species' reproductive and pollination biology, we also examined the pollination system, per-visit pollinator effectiveness, and pollen-ovule (P/O) ratio of D. muscipula. RESULTS: Plants that received supplemental prey produced more flowers than control plants. They also had a higher overall fitness estimate (number of flowers × fruit set (total fruits/total flowers) × seeds per fruit), although this benefit was significant only when prey supplementation occurred in the previous growing season. Neither pollen supplementation nor the interaction between pollen and prey supplementation significantly affected overall plant fitness. CONCLUSIONS: This study reinforces the reliance of D. muscipula on adequate prey capture for flower, fruit, and seed production and a mobile pollen vector for reproduction, indicating the importance of considering insects as part of an effective conservation management plan for this species.

Potential of Silver Nanoparticles in Overcoming the Intrinsic Resistance of Pseudomonas aeruginosa to Secondary Metabolites from Carnivorous Plants.

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL-1) and only weakly sensitive to their mixtures, i.e., extracts and extracts' fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.

Photosynthetic cyclic electron transport provides ATP for homeostasis during trap closure in Dionaea muscipula.

BACKGROUND AND AIMS: The processes connected with prey capture and the early consumption of prey by carnivorous Dionaea muscipula require high amounts of energy. The aim of the present study was to identify processes involved in flytrap energy provision and ATP homeostasis under these conditions. METHODS: We determined photosynthetic CO2 uptake and chlorophyll fluorescence as well as the dynamics of ATP contents in the snap traps upon closure with and without prey. KEY RESULTS: The results indicate that upon prey capture, a transient switch from linear to cyclic electron transport mediates a support of ATP homeostasis. Beyond 4 h after prey capture, prey resources contribute to the traps' ATP pool and, 24 h after prey capture, export of prey-derived resources to other plant organs may become preferential and causes a decline in ATP contents. CONCLUSIONS: Apparently, the energy demand of the flytrap for prey digestion and nutrient mining builds on both internal and prey-derived resources.

Anaesthesia with diethyl ether impairs jasmonate signalling in the carnivorous plant Venus flytrap (Dionaea muscipula).

BACKGROUND AND AIMS: General anaesthetics are compounds that induce loss of responsiveness to environmental stimuli in animals and humans. The primary site of action of general anaesthetics is the nervous system, where anaesthetics inhibit neuronal transmission. Although plants do not have neurons, they generate electrical signals in response to biotic and abiotic stresses. Here, we investigated the effect of the general volatile anaesthetic diethyl ether on the ability to sense potential prey or herbivore attacks in the carnivorous plant Venus flytrap (Dionaea muscipula). METHODS: We monitored trap movement, electrical signalling, phytohormone accumulation and gene expression in response to the mechanical stimulation of trigger hairs and wounding under diethyl ether treatment. KEY RESULTS: Diethyl ether completely inhibited the generation of action potentials and trap closing reactions, which were easily and rapidly restored when the anaesthetic was removed. Diethyl ether also inhibited the later response: jasmonic acid (JA) accumulation and expression of JA-responsive genes (cysteine protease dionain and type I chitinase). However, external application of JA bypassed the inhibited action potentials and restored gene expression under diethyl ether anaesthesia, indicating that downstream reactions from JA are not inhibited. CONCLUSIONS: The Venus flytrap cannot sense prey or a herbivore attack under diethyl ether treatment caused by inhibited action potentials, and the JA signalling pathway as a consequence.

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells.

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin's pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl- fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

Kinematics Governing Mechanotransduction in the Sensory Hair of the Venus flytrap.

Insects fall prey to the Venus flytrap (Dionaea muscipula) when they touch the sensory hairs located on the flytrap lobes, causing sudden trap closure. The mechanical stimulus imparted by the touch produces an electrical response in the sensory cells of the trigger hair. These cells are found in a constriction near the hair base, where a notch appears around the hair's periphery. There are mechanosensitive ion channels (MSCs) in the sensory cells that open due to a change in membrane tension; however, the kinematics behind this process is unclear. In this study, we investigate how the stimulus acts on the sensory cells by building a multi-scale hair model, using morphometric data obtained from µ-CT scans. We simulated a single-touch stimulus and evaluated the resulting cell wall stretch. Interestingly, the model showed that high stretch values are diverted away from the notch periphery and, instead, localized in the interior regions of the cell wall. We repeated our simulations for different cell shape variants to elucidate how the morphology influences the location of these high-stretch regions. Our results suggest that there is likely a higher mechanotransduction activity in these 'hotspots', which may provide new insights into the arrangement and functioning of MSCs in the flytrap.

Venus Flytrap Rarely Traps Its Pollinators.

Because carnivorous plants rely on arthropods as pollinators and prey, they risk consuming would-be mutualists. We examined this potential conflict in the Venus flytrap (Dionaea muscipula), whose pollinators were previously unknown. Diverse arthropods from two classes and nine orders visited flowers; 56% of visitors carried D. muscipula pollen, often mixed with pollen of coflowering species. Within this diverse, generalized community, certain bee and beetle species appear to be the most important pollinators, on the basis of their abundance, pollen load size, and pollen fidelity. Dionaea muscipula prey spanned four invertebrate classes and 11 orders; spiders, beetles, and ants were most common. At the family and species levels, few taxa were shared between traps and flowers, yielding a near-zero value of niche overlap for these potentially competing structures. Spatial separation of traps and flowers may contribute to partitioning the invertebrate community between nutritional and reproductive functions in D. muscipula.

Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

The carnivorous Venus flytrap uses prey-derived amino acid carbon to fuel respiration.

The present study was performed to elucidate the fate of carbon (C) and nitrogen (N) derived from protein of prey caught by carnivorous Dionaea muscipula. For this, traps were fed 13 C/15 N-glutamine (Gln). The release of 13 CO2 was continuously monitored by isotope ratio infrared spectrometry. After 46 h, the allocation of C and N label into different organs was determined and tissues were subjected to metabolome, proteome and transcriptome analyses. Nitrogen of Gln fed was already separated from its C skeleton in the decomposing fluid secreted by the traps. Most of the Gln-C and Gln-N recovered inside plants were localized in fed traps. Among nonfed organs, traps were a stronger sink for Gln-C compared to Gln-N, and roots were a stronger sink for Gln-N compared to Gln-C. A significant amount of the Gln-C was respired as indicated by 13 C-CO2 emission, enhanced levels of metabolites of respiratory Gln degradation and increased abundance of proteins of respiratory processes. Transcription analyses revealed constitutive expression of enzymes involved in Gln metabolism in traps. It appears that prey not only provides building blocks of cellular constituents of carnivorous Dionaea muscipula, but also is used for energy generation by respiratory amino acid degradation.

Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.

Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin.

The Venus flytrap attracts insects by the release of volatile organic compounds.

Does Dionaea muscipula, the Venus flytrap, use a particular mechanism to attract animal prey? This question was raised by Charles Darwin 140 years ago, but it remains unanswered. This study tested the hypothesis that Dionaea releases volatile organic compounds (VOCs) to allure prey insects. For this purpose, olfactory choice bioassays were performed to elucidate if Dionaea attracts Drosophila melanogaster. The VOCs emitted by the plant were further analysed by GC-MS and proton transfer reaction-mass spectrometry (PTR-MS). The bioassays documented that Drosophila was strongly attracted by the carnivorous plant. Over 60 VOCs, including terpenes, benzenoids, and aliphatics, were emitted by Dionaea, predominantly in the light. This work further tested whether attraction of animal prey is affected by the nutritional status of the plant. For this purpose, Dionaea plants were fed with insect biomass to improve plant N status. However, although such feeding altered the VOC emission pattern by reducing terpene release, the attraction of Drosophila was not affected. From these results it is concluded that Dionaea attracts insects on the basis of food smell mimicry because the scent released has strong similarity to the bouquet of fruits and plant flowers. Such a volatile blend is emitted to attract insects searching for food to visit the deadly capture organ of the Venus flytrap.

DYSCALCULIA, a Venus flytrap mutant without the ability to count action potentials.

The Venus flytrap Dionaea muscipula estimates prey nutrient content by counting trigger hair contacts initiating action potentials (APs) and calcium waves traveling all over the trap.1,2,3 A first AP is associated with a subcritical rise in cytosolic calcium concentration, but when the second AP arrives in time, calcium levels pass the threshold required for fast trap closure. Consequently, memory function and decision-making are timed via a calcium clock.3,4 For higher numbers of APs elicited by the struggling prey, the Ca2+ clock connects to the networks governed by the touch hormone jasmonic acid (JA), which initiates slow, hermetic trap sealing and mining of the animal food stock.5 Two distinct phases of trap closure can be distinguished within Dionaea's hunting cycle: (1) very fast trap snapping requiring two APs and crossing of a critical cytosolic Ca2+ level and (2) JA-dependent slow trap sealing and prey processing induced by more than five APs. The Dionaea mutant DYSC is still able to fire touch-induced APs but does not snap close its traps and fails to enter the hunting cycle after prolonged mechanostimulation. Transcriptomic analyses revealed that upon trigger hair touch/AP stimulation, activation of calcium signaling is largely suppressed in DYSC traps. The observation that external JA application restored hunting cycle progression together with the DYSC phenotype and its transcriptional landscape indicates that DYSC cannot properly read, count, and decode touch/AP-induced calcium signals that are key in prey capture and processing.

The Adjustment Strategy of Venus Flytrap Photosynthetic Apparatus to UV-A Radiation.

The objective of this study was to investigate the response of the photosynthetic apparatus of the Venus flytrap (Dionaea muscipula J. Ellis) to UV-A radiation stress as well as the role of selected secondary metabolites in this process. Plants were subjected to 24 h UV-A treatment. Subsequently, chl a fluorescence and gas exchange were measured in living plants. On the collected material, analyses of the photosynthetic pigments and photosynthetic apparatus proteins content, as well as the contents and activity of selected antioxidants, were performed. Measurements and analyses were carried out immediately after the stress treatment (UV plants) and another 24 h after the termination of UV-A exposure (recovery plants). UV plants showed no changes in the structure and function of their photosynthetic apparatus and increased contents and activities of some antioxidants, which led to efficient CO2 carboxylation, while, in recovery plants, a disruption of electron flow was observed, resulting in lower photosynthesis efficiency. Our results revealed that D. muscipula plants underwent two phases of adjustment to UV-A radiation. The first was a regulatory phase related to the exploitation of available mechanisms to prevent the over-reduction of PSII RC. In addition, UV plants increased the accumulation of plumbagin as a potential component of a protective mechanism against the disruption of redox homeostasis. The second was an acclimatization phase initiated after the running down of the regulatory process and decrease in photosynthesis efficiency.

A unique inventory of ion transporters poises the Venus flytrap to fast-propagating action potentials and calcium waves.

Since the 19th century, it has been known that the carnivorous Venus flytrap is electrically excitable. Nevertheless, the mechanism and the molecular entities of the flytrap action potential (AP) remain unknown. When entering the electrically excitable stage, the trap expressed a characteristic inventory of ion transporters, among which the increase in glutamate receptor GLR3.6 RNA was most pronounced. Trigger hair stimulation or glutamate application evoked an AP and a cytoplasmic Ca2+ transient that both propagated at the same speed from the site of induction along the entire trap lobe surface. A priming Ca2+ moiety entering the cytoplasm in the context of the AP was further potentiated by an organelle-localized calcium-induced calcium release (CICR)-like system prolonging the Ca2+ signal. While the Ca2+ transient persisted, SKOR K+ channels and AHA H+-ATPases repolarized the AP already. By counting the number of APs and long-lasting Ca2+ transients, the trap directs the different steps in the carnivorous plant's hunting cycle. VIDEO ABSTRACT.

Stretch-activated ion channels identified in the touch-sensitive structures of carnivorous Droseraceae plants.

In response to touch, some carnivorous plants such as the Venus flytrap have evolved spectacular movements to capture animals for nutrient acquisition. However, the molecules that confer this sensitivity remain unknown. We used comparative transcriptomics to show that expression of three genes encoding homologs of the MscS-Like (MSL) and OSCA/TMEM63 family of mechanosensitive ion channels are localized to touch-sensitive trigger hairs of Venus flytrap. We focus here on the candidate with the most enriched expression in trigger hairs, the MSL homolog FLYCATCHER1 (FLYC1). We show that FLYC1 transcripts are localized to mechanosensory cells within the trigger hair, transfecting FLYC1 induces chloride-permeable stretch-activated currents in naïve cells, and transcripts coding for FLYC1 homologs are expressed in touch-sensing cells of Cape sundew, a related carnivorous plant of the Droseraceae family. Our data suggest that the mechanism of prey recognition in carnivorous Droseraceae evolved by co-opting ancestral mechanosensitive ion channels to sense touch.

Mechanical factors contributing to the Venus flytrap's rate-dependent response to stimuli.

The sensory hairs of the Venus flytrap (Dionaea muscipula Ellis) detect mechanical stimuli imparted by their prey and fire bursts of electrical signals called action potentials (APs). APs are elicited when the hairs are sufficiently stimulated and two consecutive APs can trigger closure of the trap. Earlier experiments have identified thresholds for the relevant stimulus parameters, namely the angular displacement [Formula: see text] and angular velocity [Formula: see text]. However, these experiments could not trace the deformation of the trigger hair's sensory cells, which are known to transduce the mechanical stimulus. To understand the kinematics at the cellular level, we investigate the role of two relevant mechanical phenomena: viscoelasticity and intercellular fluid transport using a multi-scale numerical model of the sensory hair. We hypothesize that the combined influence of these two phenomena and [Formula: see text] contribute to the flytrap's rate-dependent response to stimuli. In this study, we firstly perform sustained deflection tests on the hair to estimate the viscoelastic material properties of the tissue. Thereafter, through simulations of hair deflection tests at different loading rates, we were able to establish a multi-scale kinematic link between [Formula: see text] and the cell wall stretch [Formula: see text]. Furthermore, we find that the rate at which [Formula: see text] evolves during a stimulus is also proportional to [Formula: see text]. This suggests that mechanosensitive ion channels, expected to be stretch-activated and localized in the plasma membrane of the sensory cells, could be additionally sensitive to the rate at which stretch is applied.

An approach to the plant body: Assessing concrete and abstract aspects.

The paper aims at proposing a representation of plants as individuals. The first section selects the population of plants to which this study is addressed. The second section describes the effective architecture of plants as modular systems with fixed and mobile elements, in other words, plants and their extensions. The third section presents how plants integrate the fixed and mobile modules into functional units through three areas of particular relevance to plant growth and development: nutrition, defence and pollination. Based on the tangible elements introduced in the previous sections, the fourth section presents the main issue of the proposal which is not apparent at first glance, namely, the local-global relationship in plants' architecture that determines their individuality as organisms. Finally, in the conclusion, we issue the challenge of developing a collective presentation of plants which satisfies their complementary dimension.

Is a blue-red light a good elicitor of phenolic compounds in the family Droseraceae? A comparative study.

Plants from the family Droseraceae, especially Drosera sp. and Dionaea sp., are naturally rich in phenolic derivatives such as plumbagin, among others. Plumbagin is known both for its pharmacological significance and its protective properties against light stress. Light stress - high light intensity or/and light spectral composition - activates plants' response mechanisms including, among others, hormonal (salicylic acid, jasmonic acid) pathways and secondary metabolite (phenolic compounds, proline) pathways. Short-wavelength radiation, due to its high energy, will induce the synthesis of protective secondary metabolites, including those with pharmaceutical properties. The aim of the study was to describe and compare acclimation strategies of Drosera peltata and Dionaea muscipula to blue-red light in the context of phenolic compound accumulation, and salicylic acid, jasmonic acid and proline synthesis. For the first time, differences in the responses of D. muscipula and D. peltata to blue-red light (in the ratio 6:1) were established. In Dionaea sp., it was associated with the use of redox equivalents (in particular, plastoquinone pool) for the synthesis of primary metabolites used in the process of growth and development. In Drosera sp., a rapid adjustment of redox state led to the synthesis of secondary metabolites, constituting a reservoir of carbon skeletons and allowing for a quick defence response to stress factors. In both species, blue-red light did not induce the jasmonic acid pathway. However, the salicylic acid pathway was induced as an alternative to the phenolic compound synthesis pathway. Nevertheless, the applied blue-red light was not an effective elicitor of phenolic compounds in the plants examined.

Signaling in electrical networks of the Venus flytrap (Dionaea muscipula Ellis).

The Venus flytrap captures insects with one of the most rapid movements in the plant kingdom. There is a significant difference between properties of electrical signals generated in the Venus flytrap described in literature. Amplitudes of action potentials vary from 14 mV to 200 mV with duration of signals from 2 ms to 10 s. Here we present experimental study of potential differences between Ag/AgCl electrodes inserted to the trap, petiole, and into soil or external ECG electrodes attached to surfaces of the Venus flytrap. Diverse types of electrodes with various positions in a plant tissue or in soil show different amplitude and duration of electrical signals because potentials are measured in different electrochemical circuits. Electrical signals in the Venus flytrap were induced by mechanical stimulation of the trigger hairs or by chemical stimulation of a midrib using small drops of H2O2 or HNO3. Here we found that action potentials can propagate with speed up to 10 m/s in the trap of D. muscipula. Results are compared with equivalent electrical circuits.

Plasma-generated reactive oxygen and nitrogen species can lead to closure, locking and constriction of the Dionaea muscipula Ellis trap.

Reactive oxygen and nitrogen species (RONS) can influence plant signalling, physiology and development. We have previously observed that an argon plasma jet in atmospheric air can activate plant movements and morphing structures in the Venus flytrap and Mimosa pudica similar to stimulation of their mechanosensors in vivo. In this paper, we found that the Venus flytrap can be activated by plasma jets without direct contact of plasma with the lobe, midrib or cilia. The observed effects are attributed to RONS, which are generated by argon and helium plasma jets in atmospheric air. We also found that application of H2O2 or HNO3 aqueous solutions to the midrib induces propagation of action potentials and trap closing similar to plasma effects. Control experiments showed that UV light or neutral gas flow did not induce morphing or closing of the trap. The trap closing by plasma is thus likely to be associated with the production of hydrogen peroxide by the cold plasma jet in air. Understanding plasma control of plant morphing could help design adaptive structures and bioinspired intelligent materials.