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 []. Once trapped, the still-moving victim continues to activate these mechanosensors, prolonging the electrical stimulation for many hours ( Figure 1 A). In these experiments, we recorded 63 ± 13 APs during the first hour after prey capture (n = 5, mean ± SE).

(B) Quantification of DmJAZ1 (left axis, black bars) and DmCOI1 (right axis, red bars) transcript levels in response to the number of elicited APs (as indicated). Traps were pre-treated 4 hr before application of APs with H 2 O (shaded in white) or 100 μM COR-MO (shaded in gray). Transcript numbers are given relative to 10,000 molecules of DmACT1. Asterisks indicate a statistically significant difference to zero applied APs ( ∗ p < 0.05; ∗∗ p < 0.01 by one-way ANOVA) (n ≥ 6, mean ± SE).

(A) Surface potential recordings from the surface of a Venus flytrap lobe during cricket capture. During the first hour, 51 APs were elicited, and within 6 hr of measurement, more than 100 APs were recorded. The inset (red frame, enlarged in the lower panel) shows the first seven APs that lead to prey capture and initiation of digestion. The picture shows a Dionaea plant with attached surface electrode to a trap lobe. The presented graph is representative of five individual experiments.

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

5 Escalante-Pérez M.

Krol E.

Stange A.

Geiger D.

Al-Rasheid K.A.S.

Hause B.

Neher E.

Hedrich R. A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap. 13 Vadassery J.

Reichelt M.

Jimenez-Aleman G.H.

Boland W.

Mithöfer A. Neomycin inhibition of (+)-7-iso-jasmonoyl-L-isoleucine accumulation and signaling. 14 Maffei M.

Bossi S.

Spiteller D.

Mithöfer A.

Boland W. Effects of feeding Spodoptera littoralis on lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components. 15 Dombrowski J.E.

Bergey D.R. Calcium ions enhance systemin activity and play an integral role in the wound response. 16 Fisahn J.

Herde O.

Willmitzer L.

Peña-Cortés H. Analysis of the transient increase in cytosolic Ca2+ during the action potential of higher plants with high temporal resolution: requirement of Ca2+ transients for induction of jasmonic acid biosynthesis and PINII gene expression. 5 Escalante-Pérez M.

Krol E.

Stange A.

Geiger D.

Al-Rasheid K.A.S.

Hause B.

Neher E.

Hedrich R. A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap. 6 Libiaková M.

Floková K.

Novák O.

Slováková L.

Pavlovič A. Abundance of cysteine endopeptidase dionain in digestive fluid of Venus flytrap (Dionaea muscipula Ellis) is regulated by different stimuli from prey through jasmonates. 17 Pavlovič A.

Saganová M. A novel insight into the cost-benefit model for the evolution of botanical carnivory. 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 []. There is evidence from non-carnivorous plants that increases in cytosolic calcium triggers JA biosynthesis [], 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 []. 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 1 A, 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 1 A, 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 1 B).

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.

1 Tretner C.

Huth U.

Hause B. Mechanostimulation of Medicago truncatula leads to enhanced levels of jasmonic acid. 18 Reymond P.

Weber H.

Damond M.

Farmer E.E. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. 19 Chehab E.W.

Eich E.

Braam J. Thigmomorphogenesis: a complex plant response to mechano-stimulation. 20 Pauwels L.

Goossens A. The JAZ proteins: a crucial interface in the jasmonate signaling cascade. 21 Gfeller A.

Liechti R.

Farmer E.E. Arabidopsis jasmonate signaling pathway. 22 Gimenez-Ibanez S.

Boter M.

Solano R. Novel players fine-tune plant trade-offs. 18 Reymond P.

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Farmer E.E. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. 23 Scholz S.S.

Vadassery J.

Heyer M.

Reichelt M.

Bender K.W.

Snedden W.A.

Boland W.

Mithöfer A. Mutation of the Arabidopsis calmodulin-like protein CML37 deregulates the jasmonate pathway and enhances susceptibility to herbivory. 24 Chung H.S.

Koo A.J.K.

Gao X.

Jayanty S.

Thines B.

Jones A.D.

Howe G.A. Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory. 25 Acosta I.F.

Gasperini D.

Chételat A.

Stolz S.

Santuari L.

Farmer E.E. Role of NINJA in root jasmonate signaling. 26 Pauwels L.

Morreel K.

De Witte E.

Lammertyn F.

Van Montagu M.

Boerjan W.

Inzé D.

Goossens A. Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. 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) []. 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 []. 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 1 B, 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 1 B, red bars). As in non-carnivorous Arabidopsis plants [], JAZ1 induction indicates that the JA-signaling pathway is mechanically induced.

18 Reymond P.

Weber H.

Damond M.

Farmer E.E. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. 27 Benedetti C.E.

Costa C.L.

Turcinelli S.R.

Arruda P. Differential expression of a novel gene in response to coronatine, methyl jasmonate, and wounding in the Coi1 mutant of Arabidopsis. 28 Edreva A. Pathogenesis-related proteins: research progress in the last 15 years. 29 Reymond P.

Farmer E.E. Jasmonate and salicylate as global signals for defense gene expression. 30 Schulze W.X.

Sanggaard K.W.

Kreuzer I.

Knudsen A.D.

Bemm F.

Thøgersen I.B.

Bräutigam A.

Thomsen L.R.

Schliesky S.

Dyrlund T.F.

et al. The protein composition of the digestive fluid from the venus flytrap sheds light on prey digestion mechanisms. 31 Paszota P.

Escalante-Perez M.

Thomsen L.R.

Risør M.W.

Dembski A.

Sanglas L.

Nielsen T.A.

Karring H.

Thøgersen I.B.

Hedrich R.

et al. Secreted major Venus flytrap chitinase enables digestion of Arthropod prey. 30 Schulze W.X.

Sanggaard K.W.

Kreuzer I.

Knudsen A.D.

Bemm F.

Thøgersen I.B.

Bräutigam A.

Thomsen L.R.

Schliesky S.

Dyrlund T.F.

et al. The protein composition of the digestive fluid from the venus flytrap sheds light on prey digestion mechanisms. 32 Miersch O.

Neumerkel J.

Dippe M.

Stenzel I.

Wasternack C. Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling. 33 Kitaoka N.

Matsubara T.

Sato M.

Takahashi K.

Wakuta S.

Kawaide H.

Matsui H.

Nabeta K.

Matsuura H. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate. 34 Koo A.J.

Cooke T.F.

Howe G.A. Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine. 2 O, 12-hydroxy-JA-Ile, and ethynyl-ln-Ile, two isoleucine derivatives that are inactive in floral and extra-floral nectary glands [ 35 Radhika V.

Kost C.

Boland W.

Heil M. The role of jasmonates in floral nectar secretion. 36 Heil M.

Koch T.

Hilpert A.

Fiala B.

Boland W.

Linsenmair K. Extrafloral nectar production of the ant-associated plant, Macaranga tanarius, is an induced, indirect, defensive response elicited by jasmonic acid. 37 Wasternack C.

Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. 2 O, 12-hydroxy-JA-Ile, or ethynyl-ln-Ile, induced slow trap closure, secretion (cf. [ 5 Escalante-Pérez M.

Krol E.

Stange A.

Geiger D.

Al-Rasheid K.A.S.

Hause B.

Neher E.

Hedrich R. A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap. 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 []. 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 []. 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 []. These proteins are highly abundant in the green stomach formed by the closed trap lobes []. 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 S1 A). 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 []. Therefore, as a control, we applied HO, 12-hydroxy-JA-Ile, and ethynyl-ln-Ile, two isoleucine derivatives that are inactive in floral and extra-floral nectary glands [] as well as in flower and seed development []. When sprayed on open traps, JA-Ile and COR, but not HO, 12-hydroxy-JA-Ile, or ethynyl-ln-Ile, induced slow trap closure, secretion (cf. []), and transcription of hydrolases ( Figure S1 B). These effects were consistent with the trigger-hair-based electrostimulation of the green stomach ( Figure S1 A).