The Venus Flytrap Dionaea muscipula Counts Prey-Induced Action Potentials to Induce Sodium Uptake

Carnivorous plants, such as the Venus flytrap (Dionaea muscipula), depend on an animal diet when grown in nutrient-poor soils. When an insect visits the trap and tilts the mechanosensors on the inner surface, action potentials (APs) are fired. After a moving object elicits two APs, the trap snaps shut, encaging the victim. Panicking preys repeatedly touch the trigger hairs over the subsequent hours, leading to a hermetically closed trap, which via the gland-based endocrine system is flooded by a prey-decomposing acidic enzyme cocktail. Here, we asked the question as to how many times trigger hairs have to be stimulated (e.g., now many APs are required) for the flytrap to recognize an encaged object as potential food, thus making it worthwhile activating the glands. By applying a series of trigger-hair stimulations, we found that the touch hormone jasmonic acid (JA) signaling pathway is activated after the second stimulus, while more than three APs are required to trigger an expression of genes encoding prey-degrading hydrolases, and that this expression is proportional to the number of mechanical stimulations. A decomposing animal contains a sodium load, and we have found that these sodium ions enter the capture organ via glands. We identified a flytrap sodium channel DmHKT1 as responsible for this sodium acquisition, with the number of transcripts expressed being dependent on the number of mechano-electric stimulations. Hence, the number of APs a victim triggers while trying to break out of the trap identifies the moving prey as a struggling Na⁺-rich animal and nutrition for the plant.

Video Abstract

Insect Capture Electrically Excites the Venus Flytrap

In order to investigate the Venus flytrap’s signaling cascade involved in the prey capturing and digestion, we monitored the electrical activity of a prey-catching flytrap via surface electrode measurements. For these experiments we used crickets (Acheta domesticus) 6–12 mm in length and with an average weight of 23.8 mg. It is well known that only two stimuli are sufficient for provoking fast trap closure, thereby capturing the insect [5]. Once trapped, the still-moving victim continues to activate these mechanosensors, prolonging the electrical stimulation for many hours (Figure 1A). In these experiments, we recorded 63 ± 13 APs during the first hour after prey capture (n = 5, mean ± SE).

Mechano-electric Signals Are Translated into JA Biosynthesis and Hydrolase Production

In a previous study, we showed that when a second AP quickly follows the first, the rapid trap closure is provoked, but there is no increase in the cytoplasmic calcium level in the gland cells. The subsequent AP arrival at the gland, however, elicits a calcium spike [5]. There is evidence from non-carnivorous plants that increases in cytosolic calcium triggers JA biosynthesis [13, 14, 15, 16], and, for Venus flytrap, studies have already shown that insect stimulation is associated with a 2-fold increase in jasmonates within just 30 min of prey capture [5, 6, 17]. To explore this association more fully, we sought to simulate the movements of prey in the capture organ and to record the resulting electrical response. As shown in the picture in Figure 1A, we attached a surface electrode to the trap lobes and mechanically stimulated the trigger hairs for up to 60 times per hour. To mimic the action of an insect, we simulated the situation with a captured insect by manual application of APs. Note that after the first applied AP, the trap was still open; to close the trap, the first and second stimuli needed to be consecutively applied, and both needed to elicit an AP. In the closed “prey capture” state, as with the insect in Figure 1A, we applied additional stimuli, one per minute, which could be registered as the fired signal between AP numbers 3 to 60 (cf. for 2 and 5 stimuli in Figure 1B).

Four hours after the first applied AP, we collected trap samples and monitored the expression of JA genes alongside those of the stomach hydrolases and solute transporters. To quantify the dose dependency, we used qPCR to track the association between the number of APs and the expression of the genes selected.

To analyze JA signaling in active traps, we focused on the first elements in the pathway JA receptor COI1 (CORONATINE INSENSITIVE 1) and its co-receptor JAZ1 (JASMONATE ZIM DOMAIN 1) [1, 18, 19]. JA-Ile binds to the COI1 receptor, which facilitates the formation of COI1-JAZ complexes. This leads to ubiquitination and subsequent degradation of the co-receptor JAZ1 [20, 21, 22]. We first measured the resting level of DmJAZ1 (GenBank: KT223139) in untreated (no mechanical stimulation) traps and found 341 ± 131 transcripts/10,000 DmACT, after which we stimulated the plants. This stimulation resulted in an increase in gene expression in the manner proportional to the number of trigger-associated APs. The first two applied APs, which close the trap, increased the DmJAZ1 transcript levels as much as 5.5-fold compared to unstimulated traps. With more than five APs elicited, JA gene-associated transcripts tended toward maximal levels (Figure 1B, black bars). In contrast to JAZ1, we found DmCOI1 (GenBank: KT223140) already expressed in resting traps, but after two AP stimulations, its transcription was significantly repressed (Figure 1B, red bars). As in non-carnivorous Arabidopsis plants [18, 23, 24, 25, 26], JAZ1 induction indicates that the JA-signaling pathway is mechanically induced.

Also, in the Arabidopsis system, mechanical wounding associated with herbivore foraging has been shown to induce the expression of defense genes, including a set of hydrolases [18, 27, 28, 29]. The Venus flytrap shows behavior comparable to the wounding response of Arabidopsis when the captured prey is recognized as a profitable nutrient source. A previous study has shown that mechanical stimulation of the Venus flytrap, by either an insect or the experimenter, induces gland cells to secrete a cocktail of lytic enzymes [30]. Accordingly, we have selected three hydrolases for further transcript profiling: the SAG12 (GenBank: KT223141) and SCPL49 (GenBank: KT223142) protease as well as the VF chitinase-I [31]. These proteins are highly abundant in the green stomach formed by the closed trap lobes [30]. Our present results indicate that transcription of the Dionaea hydrolases is also dependent on the number of mechano-electric signals, but with five APs necessary for significant gene expression, the tested hydrolases seem to follow the JA-signaling expression (Figure S1A). It appears that Dionaea may control the amount of lytic enzymes produced in and secreted by the gland cells via the number of electrical signals activating the JA pathway. To test this assumption, we treated the flytrap with JA-Ile, the physiologically active form of JA, and its molecular mimic COR (coronatine), in the absence of any mechano-electric stimulation. Metabolic conversion of JA-Ile to hydroxylated 12-hydroxy-JA-Ile appears to inactivate JA signaling [32, 33, 34]. Therefore, as a control, we applied H₂O, 12-hydroxy-JA-Ile, and ethynyl-ln-Ile, two isoleucine derivatives that are inactive in floral and extra-floral nectary glands [35, 36] as well as in flower and seed development [37]. When sprayed on open traps, JA-Ile and COR, but not H₂O, 12-hydroxy-JA-Ile, or ethynyl-ln-Ile, induced slow trap closure, secretion (cf. [5]), and transcription of hydrolases (Figure S1B). These effects were consistent with the trigger-hair-based electrostimulation of the green stomach (Figure S1A).

We have shown that in the flytrap, similar to other plant systems, COR is the only compound with JA-Ile agonistic activity. Recently it was shown that the COR derivative coronatine-O-methyloxime (COR-MO) reverts the effects of JA-Ile and COR treatments [38]. To dissect the JA pathway in the Dionaea system, we synthesized COR-MO and pre-treated flytraps with the JA inhibitor prior to trigger-hair stimulation. Figure 1B (shaded in gray) shows that COR-MO prevents the response of DmCOI1/DmJAZ1 and stomach hydrolases (Figure S1A) to mechano-electric stimulation. Even after the application of as many as 60 APs, no significant gene regulation was detectable when the JA inhibitor was applied (gray bars). This indicates that a touch to the trigger hair is converted into an electrical signal, which, when received at the level of the gland cells, induces prey decomposition via the JA pathway. But what are the pathways for the uptake of prey-derived solutes?

Stimulated Flytraps Take Up Prey-Derived Sodium

Humans gain the sodium ions required for electrical excitability (i.e., APs) to a large extent from a meat diet [39, 40]. In the context of flesh-eating plants, we postulated whether Dionaea takes advantage of prey-available Na⁺. If this is the case, the question is does it affect the AP? To monitor how a flytrap handles a sodium-rich meal, we monitored the movement of the positive-charged sodium ion and Na⁺-induced membrane responses of gland cells in Dionaea’s “green stomach.” For sodium uptake experiments, we used a standardized animal powder with a defined ion content (Table S1) [41]. This fodder paste with a Na⁺ concentration of ∼5 mM was applied to the inner trap surface, whereupon the sensory hairs were stimulated to close the trap and initiate digestion. By sampling the green stomach, we determined the time-dependent changes in trap sodium content by inductively coupled plasma optical emission spectroscopy (ICP-OES). We found that the level of sodium started to rise >6 hr after initiating feeding. It reached an increased steady state at 12–24 hr, lasting for at least 2–3 days (Figure 2A). This steady-state Na⁺ content of the fed trap was about twice that of control traps (at t = 0 hr) and those only fed for 6 hr. In contrast to the sodium accumulation in the trap, feeding did not increase the sodium content of the petiole significantly (Figure 2A, red bars). This indicates that Dionaea takes up and retains sodium in the capture organ. To confirm this finding and to study the sodium intake at a higher resolution, we focused on the ∼37,000 glands covering the trap’s inner surface. Each gland is engaged with the secretion of digestive fluid and uptake of nutrients [42]. Given that COR triggers processes similar to those activated by a natural prey, we used sodium-free, chemically well-defined JA mimic to study the sodium transport into glands. We stimulated gland cells with COR in the presence of 1.15 μmol sodium, the approximate load carried by a fly [11, 43]. As a result, the sodium content in Dionaea gland cells increased 7-fold after 3 days of digestion (Figure 2B).

Sodium Ions Depolarize Activated Gland Cells

To follow the Na⁺ response of the gland cell’s membrane, we used intracellular voltage-recording electrodes. Single trap lobes were fitted into a perfusion chamber, and the electrical response to Na⁺ loads was recorded (cf. [44, 45]). In the initial set of experiments, glands of traps that had not been previously stimulated by a prey were challenged with buffers containing up to 120 mM Na⁺ (approximately the sodium concentration of our blood). During the stepwise increase in extracellular Na⁺, the glands were depolarized from −116 ± 19 mV to −99 ± 6 mV and finally to −82 ± 9 mV (Figure 2C). In a complementary set of experiments, sodium was applied to traps previously stimulated by living insects (cf. [30]). When compared to non-triggered glands, those previously stimulated exhibited more pronounced Na⁺-dependent depolarizations. Such “activated” gland cells depolarized in response to 120 mM Na⁺ from −122 ± 14 mV to −41 ± 29 mV.

To elucidate the affinity of the Na⁺ transporter more precisely, we applied a Na⁺ protocol of nine steps with Na⁺ concentrations ranging from 0.3 to 120 mM, both in prey-stimulated and non-stimulated gland cells. Plotting the steady-state depolarization as a function of the substrate concentration resulted in a Na⁺ saturation curve that could be best fitted using a Michaelis-Menten function. In non-stimulated traps, a _ΔV_max at 120 mM Na⁺ of 37 mV and an apparent half maximal effective concentration (EC₅₀) of about 21 mM was derived, while in gland cells from “stomach”-forming, digesting traps, the maximum membrane depolarization at 120 mM Na⁺ was about 80 mV, and the EC₅₀ value dropped to 7 mM (Figure 2D). Similar behavior was observed with COR-treated traps. These results indicate that the plasma membrane of gland cells operates a sodium conductance that is induced upon insect or COR stimulation of the flytrap.

Flytrap Contact with Prey Induces Gland Cell Expression of a Sodium-Selective Channel

To test whether Dionaea in general and glands in particular express sodium channels, we searched a Dionaea muscipula expressed sequence tag (EST) collection [46]. Although we did not find any animal-type sodium channel sequences [47, 48], we were able to identify an ortholog of the Trk/Ktr/HKT family (Figure S2). We named this flytrap isoform DmHKT1 (GenBank: KT223138) (after the first plant study naming it HKT1 and the convention agreement in the plant field) [49].

Electrical Excitement Induces Nutrient Transporter Biosynthesis

Building on the finding that insect capture and digestion is associated with ongoing mechanical stimulation, we analyzed the expression of DmHKT1 as a function of the number of experimentally induced APs. In untreated Venus flytraps (0 APs), negligible expression (147 molecules/10,000 actin) of DmHKT1 was observed (Figure 3A). Application of up to three APs did not increase the expression substantially either. When we simulated the situation with a captured insect (cf. Figure 1A) by eliciting between 5 and 60 APs, trap expression of DmHKT1 increased up to 60-fold (Figure 3A). Thus, mechanical stimulation triggers DmHKT1 expression and Na⁺ uptake. That COR-MO prevented mechano-electric stimulation of DmHKT1 expression suggests that this process is controlled by the JA-signaling pathway (Figure 3A, red bars). This assumption is further supported by our finding of COR-induced secretion and distinct induced DmHKT1 expression in the trap, and particularly in secreting glands (Figures 3B–3D). The maximal induction of DmHKT1 expression in glands and traps was already observed 4 hr after stimulus onset (Figure 3C). To compare the change in DmHKT1 expression and amplitude of Na⁺-induced depolarization, we monitored the gland cell membrane properties and trap sodium channel mRNA side by side. We found the same time-dependent increase in both characteristics after COR treatment. The expression of DmHKT1, as well as the sodium-dependent depolarization, increased between 3.5 and 6 hr after COR treatment (Figure 3D). This finding supports the notion that prey-derived Na⁺ is taken up into the glands of Dionaea by DmHKT1. Traps that remained non-stimulated commenced neither secretion nor induction of DmHKT1 expression. In this way, DmHKT1 is particularly expressed in digesting glands, which are known to be responsible for nutrient uptake.

Besides DmHKT1, we have recently shown that the Dionaea NH₄⁺ and K⁺ transporter expression is also induced by the jasmonate mimic COR [44, 45]. This suggests that the expression of the nutrient-relevant transporter genes is regulated by the mechanically induced JA-signaling pathway. To test this hypothesis, we treated the trap samples with 0 or 60 mechano-electrical stimuli and analyzed DmAMT1 and DmHAK5 transcripts (black bars, Figure S1D). In line with our assumption, the concentration of NH₄⁺ and K⁺ transporter mRNA increased upon mechanical stimulation and is inhibited by COR-MO (red bars).

The Flytrap-Expressed Cation Channel Is Na⁺ Selective

The results of our differential expression analysis identified the Dionaea Trk/Ktr/HKT homolog. Trk/Ktr/HKT-type transporters have been associated with potassium, sodium, and calcium uptake [50]. To test the selectivity of the gland-expressed DmHKT1, we injected its cRNA into Xenopus oocytes. DmHKT1-expressing oocytes were analyzed using the same protocols as established for Dionaea glands (Figures 2C and 2D). Initially, frog oocytes were clamped at −140 mV (the approximate resting potential of Dionaea glands) and exposed to 5 mM sodium buffer. In response to the sodium ions, inward currents of up to −4 μA were evoked in “DmHKT1 oocytes,” but not in controls (Figure 4A). Under current-clamp conditions, the inward movement of the positively charged sodium ions depolarized the oocyte plasma membrane in 100 mM NaCl by about 100 mV (Figure S3A).

To elucidate the affinity of DmHKT1 for the sodium permeation process, we increased the external Na⁺ concentration stepwise from 1 to 100 mM in oocytes expressing the Dionaea Na⁺-selective channel and monitored the change in the membrane potential (Figure S3A). By concurrently fitting the resulting Na⁺ and voltage-dependent current responses with a Michaelis-Menten equation, a K_m value of 10 mM was calculated (Figures 4B and S3B) at a membrane potential of −140 mV (Figure 2C). This affinity obtained with DmHKT1-expressing oocytes is well in line with the sodium affinity characterized in the stimulated flytrap (Figure 2D).

To quantify the kinetics of sodium transport through gland cells in planta (Figure 4C), we used the microelectrode ion flux measuring (MIFE) technique [51, 52]. We increased the extracellular Na⁺ concentration stepwise from 0 to 50 mM and followed the net flux changes. When applied to non-stimulated and non-secreting traps, no concentration-dependent flux of Na⁺ was observed. Only when traps were pre-treated by the prey surrogate COR was the elevation of the external Na⁺ level followed by a concentration-dependent influx of the sodium ion (Figures S3C and 4C).

Since captured insects are naturally rich in sodium and potassium, we sought to simulate the condition in Dionaea’s green stomach when animals are decomposed into their components by exposing DmHKT1 oocytes to both these alkali cations. In contrast to Na⁺, the application of K⁺ within the same concentration range did not evoke DmHKT1-type inward currents (Figure S3D). Notably, no DmHKT1-mediated electrical responses in the presence of sodium could be detected, even with a K⁺ concentration ten times higher than that used for Na⁺ (Figure 4D). In response to changes in the extracellular Na⁺ concentration from 1 to 10 and 10 to 100 mM, the reversal potential of the sodium current shifted by 54.7 mV (±0.94 mV) and 55.3 mV (±0.94 mV), respectively, in the heterologous Xenopus oocytes expression system (Figure S3E). This near-Nernstian behavior of the reversal potential provides biophysical evidence for the operation of DmHKT1 as an ion channel, like its bacterial homolog TrkH [53, 54]. This notion was further confirmed by determining the activation energy (Figure S3F). The Q₁₀ value is a unit-less quantity that distinguishes Nernstian-type ion channel transport events from carrier-mediated ones on the basis of their activation energy. Changes in the temperature profile only weakly affected DmHKT1-dependent Na⁺ currents (Figure S3F), and the Q₁₀ value of 1.27 ± 0.01, derived from Arrhenius plotting, clearly classifies the gland cell sodium transport activity as channel-like [44, 45].

In line with the Na⁺ selectivity of DmHKT1, sodium exposure of excitable gland cells changed neither the amplitude nor the shape of the AP, excluding Na⁺ as a charge carrier of the Dionaea AP [53]. In other words, the consumption of sodium-rich food appears not to affect the AP-based information management of the flytrap.

How Is an Appropriate Cost-Benefit Balance Maintained during Insect Capture?

Carnivorous plants that depend on animal food should compensate or even exceed the cost of their carnivorous life cycle. Such expenses include trap generation, maintenance, and prey processing, and these must be balanced by the energy gained from the captured animal. To prevent any imbalance (i.e., net energy loss), Dionaea controls the processes in its capture organ via trap-specific mechano-electric and touch hormonal signaling. In this system, occasional single trigger-hair stimulation is recognized as subthreshold signal and memorized; it does not provide sufficient information to justify closing the trap. In contrast, two mechano-electric signals received within about 30 s of each other are judged sufficient to close the trap. The fast biomechanics of closure are now well understood, but not the cellular mechanisms that induce the destabilization of the metastable, open trap [55, 56, 57]. In this work, we did not focus on the molecular events involved in fast trap closure but the subsequent endocrine processes in the glands. Activating the glands to produce and secrete nitrogen-rich digestive enzymes is inherently costly; thus, this process appears to be closely controlled. In the carnivorous plant Nepenthes, the proteolytic activity required to digest the prey seems to be induced and regulated by jasmonate [58]. When the second AP was received right after the initial one, the flytrap closed. At the same time, the electrical closing stimulus induced differential expression of JA receptor COI1 and co-receptor JAZ1. The induction of JAZ1 and the repression of DmCOI1 may be a part of a negative regulatory feedback loop. It has been shown that gland cells that receive a series of at least three APs increase their cytoplasmic calcium level [5]. A rise in calcium has been found to be associated with wounding-induced JA biosynthesis in non-carnivorous plants such as Arabidopsis [13, 14, 15, 16, 59]. An accumulation of jasmonates has been described as insect induced in the carnivorous sundew plant [7]. Thus, the mechano-electric stimulation of the green stomach via JA signaling triggers transcription of prey-degrading enzymes. Interestingly, expression of such hydrolases is coupled to that of the transporters required for nutrient uptake; prey movement, which we simulated with “5-plus-X” consecutive APs, triggers the de novo synthesis of the sodium channel DmHKT1 (Figure 3A).

Mechanical Signaling in the Flytrap

In leaves of non-carnivorous plants such as Arabidopsis, a locally applied touch stimulus triggers an increase in both cytoplasmic calcium and JA signaling in the stimulated area as well as systemically in defined, directly connected leaves [18, 19]. However, the point where touch ends and wounding starts is difficult to define, but when the stimulus results in wounding, defense genes in both the “local” leaf receiving the stimulus and in non-treated “systemic” leaves are activated [60]. The long-distance signaling of wounding by herbivores is, for example, associated with a stimulus-induced traveling electrical wave [61]. Although the knowledge regarding touch signaling in the model plant Arabidopsis is still rather fragmentary, our present data obtained from Dionaea allow us to propose the first working model of touch signaling in the flytrap.

We have demonstrated that the mechanical energy is received at the multicellular trigger hair and converted into an electrical signal, an AP. The APs originating from the trigger hair on one lobe travel through the entire trap to reach two major targets: (1) the motor tissue, which initiates fast trap closure and the formation of the green stomach, and (2) the glands, the endocrine system responsible for the prolonged processing of the nutrient and sodium-rich animal meal. Pre-treating traps with COR-MO suppressed AP-induced stomach formation and the filling of the stomach with the lytic moiety. Such COR-MO traps, even when having received as many as 60 mechano-electric stimuli, reopen, similar to the reopening of untreated traps after the two APs required for the initial close. When we administered an increasing number of stimuli, we found COI1/JAZ1 expression was altered after only two APs, while to achieve a significant differential expression of the hydrolases and nutrient and sodium transporters, about five APs were required. Based on this behavior, we place the induction of genes operating in the endocrine system downstream of JA biosynthesis and signaling.

Dionaea Trap’s Na⁺ Channel Is Capable of Processing Prey-Derived Sodium Loads

We have been able to show that stimulated flytraps acquire and accumulate prey-derived sodium (Figures 2, 4C, and S3C) via a Dionaea sodium channel (DmHKT1), which operates in gland cells (Figure 3). The expression of the channel is induced during the process of prey capture and digestion, providing the gland cells with the necessary capacity to take up the animal Na⁺ released during decomposition of the insect (Figure 2A). Mechanical stimulation by prey induces HKT1 expression (Figure 3A). During this resorption phase, sodium is taken up by the glands and channeled into traps, while the sodium content in the petioles does not change (Figure 2). Thus, sodium is most likely stored in the large central vacuoles of the parenchyma cells.

Conclusions

Together, these findings suggest that a moving object is identified as a struggling Na⁺-rich animal trying to escape the trap. As a result, touch-number-induced expression and production of digestive enzymes is accompanied by an increase in the number of DmHKT1 transporters. Sodium uptake and storage in the trap parenchyma of carnivorous Dionaea is reminiscent of the salt management by the succulent-leaf-type halophytes [62, 63]. In halophyte species, Na⁺ can play a beneficial role in metabolic and osmotic functions and can be used as a “cheap osmoticum.” Furthermore, optimal growth and yield is observed in the presence of 100–200 mM NaCl in the soil solution [64, 65], and some of these plants can accumulate substantial (in excess of 1 M on a fresh weight [FW] basis) amounts of Na⁺ in their shoot tissues. Thus, assuming plants possess an efficient mechanism for vacuolar Na⁺ sequestration, a high Na⁺ content in plant tissues is not necessarily associated with toxicity or any detrimental effects. In the case of Dionaea plants that live in K⁺-depleted soils, Na⁺ could be a substitute cation that enables efficient osmotic adjustment. Thus, Dionaea appears to take advantage of Na⁺ intake for maintaining its cellular osmotic potential and turgor.

Surface Potential Measurements

To quantify APs in the Venus flytrap, we used non-invasive surface potential electrodes. One silver electrode was fixed with tape to the outer trap surface with the electrical connection improved by applying a droplet of Kontakt-Gel (Laboklinika). The reference electrode was put into the wet soil. Electrical signals were amplified 100× and recorded with PatchMaster software (HEKA).

Feeding Experiments

The sodium uptake capacity of traps was determined by feeding traps with insect powder containing defined amounts of the respective cation. Five traps per plant were fed with 20 mg insect powder, dissolved in 100 μl water. The insect powder was derived from adult moths of Vanessa cardui (Lep.: Nymphalidae) and had an approximate Na⁺ content of 5 mM. At the given time points after feeding, traps were harvested, the remaining insect material was removed, and the plant tissue was thoroughly dried. The ground plant powder was then hydrolyzed with HNO₃ in a pressure ashing device (Seif). Traps diluted with H₂O were analyzed by ICP-OES (JY 70 Plus, Devision d’Instruments S.A., Jobin Yvon), and petioles of fed traps were analyzed by flame atom absorption spectrometer (F-AAS) (AAnalyst 400, PerkinElmer).

Laser-Ablation ICP-MS

The laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been described elsewhere [66]. In brief, Dionaea traps on intact plants were treated with 1.15 μmol sodium (in 50 μl; 23 mM), coronatine 100 μM, or sodium and coronatine together for 3 days. Glands were ablated using a solid state NYAG laser (UP193 SS, New Wave Research). ¹³C and ²³Na signals were detected using the quadrupole ICP-MS (7500 CX, Agilent Technologies), with the ¹³C signal serving as the internal standard.

Cloning of DmHKT1

Searching the available expressed sequence tag (EST) data from Dionaea muscipula [30, 46] revealed a coding sequence related to HKT1-like sodium transporters. PolyA-mRNA was isolated from 10 μg of total RNA using Dynabeads (Invitrogen) according to the manufacturer’s instructions. cDNA was generated from Dionaea muscipula whole-plant mRNA. The cDNA of DmHKT1 was amplified using gene-specific oligonucleotide primers, which comprised the coding DNA sequence (CDS) of DmHKT1, and Advantage cDNA polymerase mix (Clontech). This revealed a sequence identical to that expected from the EST data. The full-length clone was inserted into pGEM-T Easy using the pGEM-T Easy Vector System I (Promega). For heterologous expression in Xenopus oocytes, the generated cDNA of DmHKT1 was cloned into oocyte expression vectors (based on pGEM vectors) using the advanced uracil-excision-based cloning technique described by Nour-Eldin et al. [67]. For functional analysis, cRNA was prepared using the mMessage mMachine T7 Transcription Kit (Ambion). For oocyte electrophysiological experiments, 25 ng of DmHKT1 cRNA was injected and expressed for 2–3 days.

Synthesis of COR-MO

COR-MO was synthesized according to Monte [38]. All chemicals used were obtained from Sigma-Aldrich. Briefly, 1 mg coronatine was dissolved in ca. 350 μl dried pyridine, and 90 mg solid O-methylhydroxylamine hydrochloride (350 molar excess) was added. The reaction mixture was incubated at 23°C for 18 hr. Non-reacted O-methylhydroxylamine hydrochloride was removed by adding 400 μl water. The product COR-MO was extracted three times from pyridine-water solution using ethyl acetate. The pooled ethyl acetate fraction was washed with water and then dried over sodium sulfate. Thereafter, the solvent was removed by desiccation in vacuo. Conformation of the product was provided by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy and compared with published spectral data [38].

Plant Material and Tissue Sampling

Dionaea muscipula plants were purchased from CRESCO Carnivora and grown in plastic pots at 22°C in a 16:8 hr light:dark photoperiod. The material for the expression analyses was harvested as follows: traps, petioles, and trap rims were separated and immediately frozen in liquid nitrogen. Additionally, secretory cells were isolated from the inner trap surface by gently abrading the gland complexes with a razor blade. For induction of secretion and the signal compound treatment, 100 μM COR or the compound solution (Sigma-Aldrich) was directly sprayed onto the respective tissues, and samples were harvested after 24 hr or at the given time points. In inhibitor tests, 100 μM COR-MO was sprayed 4 hr before mechanical stimulation was applied to the Venus flytraps. For mechanical induction of expression, trigger hairs were stimulated 1–60 times (1/min), and samples were collected 4 hr after the first stimulus. Harvested tissue was immediately frozen in liquid nitrogen.

Expression Analyses

Total RNA was separately isolated from each sample and transcribed into cDNA using M-MLV reverse transcriptase (Promega). Quantification of the actin transcript DmACT1 (GenBank: KC285589) and Dionaea transcripts was performed by real-time PCR as described elsewhere [68]. Dionaea transcripts were normalized to 10,000 molecules of DmACT1.

Non-invasive Ion Flux Measurements

Net fluxes of Na⁺ and K⁺ were measured using the non-invasive MIFE technique (University of Tasmania, Australia). The theory of non-invasive MIFE measurements and all specific details of microelectrode fabrication and calibration are available in our prior publications [51, 52, 69]. Briefly, microelectrodes with an external tip diameter of ∼2 μm were pulled, silanized and filled with an appropriate ion-selective cocktail. For K⁺ we used the commercially available cocktail from Sigma: 60031, while for Na⁺, we used a highly Na⁺-selective resin that is not responsive to K⁺ [70]. Electrodes were calibrated with an appropriate set of standards and mounted on a 3D micromanipulator (MMT-5, Narishige). An immobilized lobe was placed into a 90-mm Petri dish containing 40 ml basic salt media (BSM: 0.2 mM KCl, 0.1 mM CaCl₂, adjusted to the required pH using Mes/Tris) and placed into a Faraday cage. Na⁺- and K⁺-selective microelectrodes were positioned with their tips aligned, 50 μm above the lobe surface using a 3D hydraulic manipulator. During measurement, a computer-controlled stepper motor moved the electrodes in a slow (10 s) square-wave cycle between the two positions, close to (50 μm) and away from (150 μm) the trap surface. The potential difference between two positions was recorded by the MIFE CHART software [51] and converted to an electrochemical potential difference using the calibrated Nernst slopes of the electrodes. Net ion fluxes were calculated using the MIFEFLUX software for cylindrical diffusion geometry [71].

To study the dose dependence of Na⁺ uptake, intact Dionaea leaves were pre-treated with coronatine for 24 hr as described above. A single lobe was cut and immobilized in a measuring chamber using medical adhesive (VH355, Ulrich AG). The lobe was left for adaptation in BSM solution before flux measurements commenced. Net Na⁺ and K⁺ fluxes were measured in response to the consecutive increase in NaCl content in the external BSM (NaCl, mM: 0, 3, 10, 50 mM) for 3 min at each NaCl concentration. The pH of the media was maintained at pH 5. Leaves without coronatine treatment were used as controls.

Intracellular Measurements

Prior to measurement, the lobe of a cut trap was glued to the base of a chamber and left to recover (30 min) in a plant-standard solution containing 0.1 mM KCl, 10 mM CaCl₂, and 15 mM MES, adjusted with Tris to pH 6. Osmolarity was maintained at 240 mOsm/kg with D-sorbitol when needed. The NaCl addition to the standard solution was at mM concentrations: 0.3, 0.6, 1.2, 3, 6, 12, 30, 60, 120. During experiments, leaves were continuously perfused with the plant-standard solution (1 ml/min). For stimulation, a 1-min exposure to the respective NaCl concentration was sufficient.

Oocyte Recordings

For double-electrode voltage-clamp studies (DEVC), oocytes were perfused with Tris/Mes-based buffers. The standard bath solution contained 10 mM Tris/Mes (pH 5.6), 1 mM CaCl₂, 1 mM MgCl₂, and 1 mM LaCl₃. The osmolality of each solution was adjusted to 220 mosmol/kg using D-sorbitol. To balance the ionic strength for measurements under varying cation (Na⁺ and K⁺) concentrations, we compensated with lithium to 100 mM. Solutions for selectivity measurements were composed of the standard bath solution supplemented with 100 mM Na⁺, NMDG⁺ (N-methyl-d-glucamine), Li⁺, K⁺, Rb⁺, or Cs⁺ chloride salts. For current-voltage relations (IV-curves), single-voltage pulses were applied in 10 mV increments from −200 to +60 mV after starting from a holding potential (V_H) of 0 mV. Steady-state currents (I_SS) were extracted at the end of the test pulses lasting 50 ms. Temperature experiments were performed under standard solution conditions as described by Scherzer et al. [45]. The Q₁₀ value is proportional to the free energy of activation (_ΔG), and it is the factor by which the transport rate increases when the temperature is changed by ten degrees. The only known transport processes for small hydrophilic ions with Q₁₀ values below 2.0 are catalyzed by channel proteins. Data were analyzed using Igor Pro and Origin Pro 9.0G.

Document S1. Figures S1–S3 and Table S1

Document S2. Article plus Supplemental Information

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Supplementary Materials