Review

Venus Flytrap: How an Excitable,

Carnivorous Plant [623_TD$IF]Works
Rainer Hedrich1,* and Erwin Neher2

The carnivorous plant Dionaea possesses very sensitive mechanoreceptors. Highlights

Upon contact with prey an action potential is triggered which, via an electrical Best known from the animal kingdom,
network – comparable to the nervous system of vertebrates – rapidly closes its carnivory in the green branch of the
tree of life at a first glance seems to
bivalved trap. The ‘hunting cycle’ comprises a constitutively activated mecha- be a peculiar trait. Thanks to recent
nism for the rapid capture of prey, followed by a well-orchestrated sequence of advances, we now know that plant
carnivory evolved at least six times
activation of genes responsible for tight trap closure, digestion of the prey, and independently.
uptake of nutrients. Decisions on the step-by-step activation are based on
‘counting’ the number of stimulations of sensory organs. These remarkable Actively trapping carnivores such as
the Venus flytrap, Dionaea muscipula,
animal-like skills in the carnivore are achieved not by taking over genes from its have fascinated scientists since Dar-
prey but by modifying and rearranging the functions of genes that are ubiqui- win’s time, but the molecular basis for
the so-called carnivorous syndrome
tous in plants.
appears enigmatic.

‘Omics’ approaches have shed new

Model Plants Do Not Eat Meat light on the molecular events accom-
Charles Darwin observed that the Venus flytrap Dionaea muscipula lives on an animal diet, and panying individual steps in the Venus
flytrap hunting cycle, suggesting that
called it ‘the most wonderful plant in the world’ [1]. Carnivorous plants, such as Dionaea, have
carnivory originates from a defense
climbed the Darwinian ladder and have turned from prey to predator [2]. Darwin in his time mechanism.
demonstrated that Dionaea abruptly closes its trap and imprisons the victim when the prey
strikes its sensory hairs. The fact that the Venus flytrap can catch animals has fascinated not Although the genomic tools still do not
compare to those of model plants, the
only scientists but has also created fear. For instance, the mystery about a green flesh-eater is a ‘carnivorome’ established recently for
topic in the movie Little Shop of Horrors. Dionaea has helped to elucidate the
origin of plant mechanical and electri-
Although the Venus flytrap has been known for a long time, the molecular basis of the cal excitability, the coupling of electri-
cal and chemical signaling, and the
carnivorous lifestyle remained largely unexplored until recently. While Darwin based his con- molecular basis of chemosensing.
clusions exclusively on observations, nowadays we have the option to perform molecular
analyses to gain new insights into the secrets of the Venus flytrap. However, in concentrating on
an organism with specialized adaptations we are facing difficulties that certainly come with
plants for which there are no mutants and no sequence information, and where neither
transformation techniques nor genetics are available.

Therefore, when summarizing our results on Dionaea obtained over several years, we also ask – 1
Institute for Molecular Plant
Physiology and Biophysics, Julius-
what are the problems when studying non-model plants? Are they outweighed by insights into
von-Sachs Platz 2, 97082 Würzburg,
specialized mechanisms and plant biodiversity [3]? Do we need to study non-model plants to Germany
find out how plants conquered land? How can we understand how some plants can survive 2
Department for Membrane
Biophysics, Max Planck Institute for
extreme habitats or episodes of severe stress, and – in case of our research – how a plant
Biophysical Chemistry, 37077
‘hunts animals’ [1], if these skills do not exist in arabidopsis (A. thaliana)? Göttingen, Germany

To address open questions about the molecular underpinnings of the lifestyle of the green flesh-
*Correspondence:
eater, we review here insights emerging from a combination of molecular physiological,
hedrich@botanik.uni-wuerzburg.de
electrophysiological, and bioinformatic analyses. (R. Hedrich).

220 Trends in Plant Science, March 2018, Vol. 23, No. 3 https://doi.org/10.1016/j.tplants.2017.12.004
© 2017 Elsevier Ltd. All rights reserved.
The Hunting Cycle of Dionaea
Unique morphological features enable the trap-forming leaf tip of the Venus flytrap to catch and
digest prey, and subsequently to absorb prey-derived nutrients. To gain mechanistic insights
into the molecular processes underlying the carnivory syndrome, we combined ultrastructural,
physiological, and proteomic analyses with thorough transcriptome sequencing to analyze the
changes in gross gene expression patterns which occur during different steps of the hunting
cycle (Figure 1). Because one would expect that these functional properties of the trap are
based on the occurrence and abundance of specific transcript types, a reference transcriptome
of the Venus flytrap was generated [4]. This dataset provided molecular insights into the
different stages of the Dionaea hunting cycle.

Dionaea muscipula, when starving, develops intensely red-colored inner traps [5] to attract
insects. Darwin previously raised the question of whether or not the carnivore displays further
mechanisms to lure small animals in addition to trap color [1]. Plants can attract insects for

20 mV
2s

Reopening
2 APs
threshold
Fast closure
Lyc enzymes 0.1 s
HCI Nutrients
GSH

> 2 APs
JA biosynthesis/
signaling
Digeson
Absorpon Stomach formaon
12 h–5 days Secreon
1–2 h
∞ APs > 5 APs
Hydrolase/transporter Hydrolase/transporter
expression ongoing expression

Figure 1. The Hunting Cycle of Dionaea muscipula. The first mechano-electric stimulation of the trigger hair by a
trap-visiting insect sets the trap in a ‘poised to capture’ mode. One touch-induced action potential (AP) is memorized by
the trap but is insufficient for trap closure. A second AP elicited within a given period (ca 30 s) is required for fast closure
and prey capture. When trying to escape, a prey insect repeatedly touches the mechanosensors, thereby eliciting
repetitive firing of APs. Three or more APs activate the JA signaling pathway, and the capture organ becomes hermetically
sealed. Glands covering the inner surface of the stomach start to express genes encoding hydrolase enzymes that
decompose the prey into its nutrient building blocks, along with the expression of transporters for the uptake of prey-
derived nutrients. In the latter processes, mechano-electric stimulation can be replaced by direct jasmonic acid (JA)
hormone administration. The more often the trigger hairs are stimulated, APs are fired, and touch hormone is synthesized,
the longer and greater the activity of the flytrap endocrine system, a process further stimulated by ‘prey-derived molecular
patterns’. By these means the number of APs informs the plant about the size and nutrient content of the struggling prey.

Trends in Plant Science, March 2018, Vol. 23, No. 3 221

 40 mV

Depolarizaon 10 s
Repolarizaon

Resng
−120 mV Recovery

Hyperpolarizaon
[Ca2+]cyt

5 Gene
Threshold
4 expression
3
2
Threshold
Closure
1

Time

40
mV
10 minutes

Stomach formaon

2+
Figure 2. A Ca Clock Provides a Molecular Counter. Sensory cells in the hinge region of the trigger hair (top left)
convert mechanosensation into an electrical signal. Trigger hair displacement results in a typical all-or-nothing action
potential (AP, top right) together with cytosolic Ca2+[621_TD$IF] transients. This Ca2+ clock (middle) provides the molecular basis for
‘counting’. Two prey-evoked APs (arrow) trigger trap closure, while repetitive APs (bottom) drive the Ca2+ clock across
defined threshold levels to control progression through individual steps of the hunting cycle. Abbreviation: cyt,
cytoplasmic.

pollination by releasing volatile organic compounds (VOCs) from their flowers. The traps of
starving Dionaea plants emit a rich bouquet composed of more than 60 VOCs, the majority
being typical constituents of fruit and flower scents in other plants [6]. Olfactory-choice bio-
assays demonstrated that the VOCs released by the Venus flytrap strongly attract starved flies,
thus indicating that Dionaea takes advantage of this evolutionarily conserved mode of plant–
insect interaction to lure its animal prey. While exploring the inner trap lobes, attracted insect
visitors accidentally displace sensory trigger hairs, causing the firing of individual action
potentials (APs; Figure 2, top) and rapid trap closure. Ongoing electrical stimulation causes
the capture organ to become hermetically sealed, giving the victim no chance of subsequent
escape.

Final sealing of the closed trap and formation of the ‘green stomach’ for prey digestion depend
on the touch hormone jasmonic acid (JA) [7–10]. JA activates the endocrine system of the

222 Trends in Plant Science, March 2018, Vol. 23, No. 3

 4 pA
15 minutes

GSH
HCI
Lyc enzymes

Figure 3. Quantal Occurrence of Gland Secretory Events. Schematic view of the typical three-layered organization
of secretory glands covering the inner trap surface (left). Upon activation, exocytosis from acid- and enzyme-containing
secretory vesicles is induced in outer-layer L1 cells (right) that can be monitored as quantal events in amperometric
measurements (upper right; for details see text).

carnivore by inducing the expression and exocytosis of hydrolytic enzymes in the numerous
glands that cover the inner surface of the stomach (Figures 1 [625_TD$IF]and 3 ). At this stage, hydrolases
secreted into the stomach decompose the prey into its nutrient building blocks. In parallel, the
expression of transporters for the uptake of prey-derived nutrients is upregulated in glands.
Transporters are synthesized and operate as solute shuttles in gland cells (see below). The
more often the trigger hairs are touched, APs are fired, and touch hormone is synthesized, the
longer and greater the activity of the flytrap endocrine system. In this way, the number of APs
informs the plant about the size and nutrient content of the struggling prey. When, after
approximately 1 week, prey decomposition and nutrient absorption have been completed,
the trap opens again and is prepared for entry into a forthcoming hunting cycle [4,11].

What Generates the APs, and What Type of Electrical Network Operates in
Dionaea?
Neuronal networks in animals are composed of nerve cells interconnected by axons and
synapses. Higher plants do not possess specialized nerve cells with axons but, similarly to
animals, they are capable of long-distance electrical signaling [12,13]. Electrical wiring and
signaling, including the ability of Dionaea to ‘count’ (see below), relies on the existence of an
electrical circuit. Compared to even a small or ‘primitive’ brain, the structure of the flytrap
appears to be relatively simple. The two lobes of the trap contain a robust, well-ordered
second- and third-order venation grid connected by a major vein [14]. In plants, the phloem (a
network of interconnected cells) inside the veins can be considered as a ‘green cable’ that
allows the transmission of APs induced by stimulation of, for example, touch receptors [12].
This way, the phloem network conducts long-distance electrical signals possibly carried by
voltage-dependent plant-specific ion channels.

Ion Channels as the Basis of the Plant AP

The generation and propagation of an AP depends on permeability changes in the membrane
and in the conductivity of both the cytoplasm and the extracellular space [15–17]. The
conductive cytoplasm of an axon is dominated by a 100 mM K+[624_TD$IF]-based electrolyte that is
shielded by the plasma membrane from an extracellular Na+ electrolyte of similar ionic strength.
Thus, the inward Na+ and outward-directed K+ gradients constitute the driving force for the
sodium- and potassium-based AP in animals [18]. Opening of voltage-activated sodium

Trends in Plant Science, March 2018, Vol. 23, No. 3 223

channels depolarizes the membrane, whereas the activity of a variety of voltage-dependent K+
channels repolarizes it. Exactly as for the axon interior, the cytoplasm of a phloem sieve tube is
dominated by an electrolyte of around 100 mM K+, but the extracellular K+ is only about 1 mM.
In plant genomic databases, including that of the carnivorous Dionaea, Shaker-type K+
channels have been identified [19]. Some of these have been functionally characterized
[20–23]. However, they are unlikely to drive APs in combination with Na+ channels because
no animal-type Na+ channels have been found in plants [12]. In contrast to the animal system,
anion gradients at the plant plasma membrane are outwardly directed, and therefore the
opening of anion channels will depolarize the membrane [24].

Similarly to the situation in animal cells, Ca2+ gradients in plants are directed towards the
cytoplasm and are several orders of magnitude in extent [16,25,26]. However, classical
voltage-dependent Ca2+ channels are also missing in plant genomes. Could then transient
changes in Ca2+ concentration trigger Ca2+-dependent channels? In animal cells a variety of
Ca2+-triggered ion channels with different ion selectivities are known. Over the past two
decades voltage- and Ca2+-activated cation and anion channels in plants have been identified
[20,27–29]. When they open, anion channels will depolarize the plasma membrane. This
qualifies them as drivers of excitation. It will be interesting to reconstitute the Dionaea AP
by expressing chloride, Ca2+, and potassium channels from the flytrap in non-excitable plant or
animal cells in future studies.

What Do the Flytrap and Nerve-Muscle APs Have in Common, and What Are
the Differences?
When a nerve cell is depolarized from a resting level of 60 mV, APs are fired that peak at
+40 mV [18]. By contrast, Dionaea flytrap cells rest at about 120 mV, that is about 60 mV
more negative than the membrane potential of animals. When the flytrap membrane potential
depolarizes and approaches 100 mV, APs are elicited which reach about 20 mV. These
APs propagate at a velocity in the low cm
s 1[62_TD$IF] range (5–25 cm
s 1) [30–33], while in nerves they
propagate with a speed of 0.1–100 m
s 1[627_TD$IF] [16,34].

Furthermore, the Dionaea APs last about 2 s and can be fired every 2 s (0.5 Hz) [35]. By
contrast, in mammalian nerve cells AP duration is in the low millisecond range, and neurons can
fire at frequencies of up to several kHz [36]. What causes the difference?

The high speed of propagation of nerve APs is due to the fact that local depolarization rapidly
spreads to neighboring regions of the nerve fiber, and needs to depolarize the neighbor only
minimally to activate voltage-dependent Na+ channels. These are present at high density and
can respond to the voltage change in fractions of a millisecond. The relative slowness of
propagation in the phloem is probably due to the larger activation threshold of chloride
channels, their lower density, and their slow intrinsic activation kinetics. The speed of propa-
gation of APs in mammalian nerve fibers is further enhanced by myelination, which causes the
excitation to rapidly jump from one node of Ranvier to the next. By contrast, propagation in the
phloem may be hindered by cell–cell junctions [12].

The maximum frequency of response, a function of the refractory period of the nerve cell AP,
also depends on the kinetics of activation and recovery from inactivation of both Na+ and K+
channels [37]. Depending on the extracellular K+ concentration in plants, the Shaker-type
outward rectifier has a half-time activation in the 0.1–1 s range [38,39]. In the flytrap, restoration
of the pre-excitation state requires input from other sources of current. The hyperpolarizing
activity of the voltage-dependent plasma-membrane proton pump turned out to be one such

224 Trends in Plant Science, March 2018, Vol. 23, No. 3

source [40,41]. Its impact is seen as a transiently more negative membrane potential following
an AP (Figure 2 top).

Would Arabidopsis Be a Suitable Model To Study Electrical Excitability in

Plants?
All plants can sense touch, including arabidopsis [42]. Why not study haptoelectrics in the
model plant? In contrast to the force that is exerted by a fly or an ant to bend the flytrap trigger
hairs (discussed below) and to elicit an AP, much higher forces are necessary in arabidopsis to
induce an electrical wave. Furthermore, these signals in arabidopsis do not represent all-or-
nothing APs, as observed in the flytrap, but show up as longlasting potential changes that are
often irregular in shape [43]. Thus, flytrap APs are more similar to those of electrically excitable
animal cells.

It seems that the strong evolutionary drive for speed of response and economy in the use of
resources (see below) has optimized a mechanism, which exists in other plants, that serves
many different purposes. Likewise, in the animal kingdom an extreme need for speed resulted
in the giant axon of the squid. In Dionaea, speed is necessary to effectively catch prey; in the
squid it is the escape response that is optimized. Evidently, studying the nerve AP in the squid
resulted in a breakthrough in our current understanding of neuronal mechanisms – a valuable
precedent for guiding research on plant signaling.

Count, Memorize, and Decide

The ability of touch-sensing and ‘counting’ interactions with other creatures is ubiquitous in the
animal kingdom. It involves memorizing, rule-following, and results in decision-making. Special
‘number neurons’ in dedicated neuronal networks of the brain fulfill these tasks [44]. The roots
of ‘counting’ can be traced down the animal tree of life. A completely different picture, however,
emerges when asking about the numerical competence of plants. Using the Venus flytrap as
experimental system, Darwin has shown that plants have invaded the animal stronghold of
numerical competence – trap closure was only induced by two consecutive touch events [1].
This plant is not only dangerous to animals; it can even ‘count’ [35,45,46].

When an insect visits the Dionaea trap and tilts the mechanosensors on the inner surface, APs
are fired [20] (https://www.youtube.com/watch?v=aiDskGkeqzo). Our studies have shown
that, within the different steps of the hunting cycle (Figure 1), Dionaea ‘memorizes’ and ‘counts’
the number of APs to estimate the size and nutrient content of struggling prey.

Flytraps Count to ‘Two’ and Beyond To Keep a Proper Cost–Benefit

Balance
In carnivorous plants that must survive on scarce resources and depend on animal food, the
fitness cost for prey capture and processing should not exceed the energy gained by the
captured prey. To prevent a possible imbalance between cost and benefit, Dionaea tightly
controls the processes associated with prey capture and processing via trap-specific
mechano-electric and touch hormone signaling [47]. In this system an occasional single trigger
hair deflection is regarded as a false positive, whereas two mechano-electric signals perceived
within about 30 s close the trap. The latter rapid process is well understood in terms of its
biomechanics [48,49]. The underlying cellular turgor-driven process that induces the destabili-
zation of a metastable open trap [7], however, awaits further exploration. When non-carnivo-
rous plant systems perceive mechanical or electrical signals, transient rises in cytoplasmic Ca2+
levels are often observed [42,43]. These cellular response-inducing Ca2+ transients last for 20–
200 s. The fact that the Dionaea trap does not close when the interval between first and second

Trends in Plant Science, March 2018, Vol. 23, No. 3 225

trigger hair deflection (and between thus APs) exceeds 30 s may indicate that the first signal
only causes a subthreshold rise in cytoplasmic Ca2+. A second AP-induced rise in Ca2+,
however, will superimpose onto the initial one, thus surpassing the threshold and inducing
turgor changes that trigger fast trap biomechanics (Figure 2, middle). If the prey stayed calm for
more than 30 s after a first AP, the Ca2+ signal would decay and subsequent single APs would
not elicit trap closure. Once the trap has been closed by two APs, and no further APs are
elicited, the trap will open again after less than 12 h. However, usually the trapped animals
trying to escape will trigger a ‘virtual fireworks’ of signals (Figure 2, bottom), which tightly seals
their cage. This is because the Venus flytrap can ‘count’ to higher numbers.

Digestive Juices Start To Flow from ‘Five’

In gland cells, a pronounced rise in cytoplasmic Ca2+ level is seen with the arrival of the third AP
[7]. There is substantial evidence from non-carnivorous plants that increasing cytosolic Ca2+
triggers JA biosynthesis [50,51]. In the Venus flytrap, insect stimulation results in JA increase
after about 30 minutes. To explore the underlying mechanism, trigger hairs were mechanically
stimulated 2–60 times per hour, and trap samples were monitored for JA action [4,35].
Transcripts representative for JA biosynthesis and signaling, as well as of Dionaea hydrolases,
were found to strongly depend on the number of mechano-electric signals fired. At five or more
signals, the trap additionally activates the ‘JA clock’ including genes for digestive enzymes in all
of its 37 000 glands (Figure 4). This activation does not take place if the JA signal pathway is
suppressed artificially before mechanical stimulation [35], proving that the electrical signal is
converted into a hormone signal in the glands.

Five or more stimuli also induce the transporters that permit absorption of the digestion
products into the plant [35]. This finding may indicate that Dionaea controls the amount of
lytic enzymes that are produced and secreted by the gland cells, as well as nutrient uptake, in a
JA-dependent manner by counting the number of electrical signals. Counting and trigger hair-
based electrostimulation precedes the formation of the green stomach (Figure 1) because
jasmonates sprayed on open traps were found to be sufficient to initiate the process. Likewise,

Fast trap
closure
Stomach
formaon

Ca2+ JA
clock clock Hydrolase
expression

Transporter
expression

2+
Figure 4. An Interconnected Ca /Jasmonate Clock Drives Flytrap Decision-Making. The Ca2+ clock triggers
fast trap closure when crossing the first threshold (two action potentials, APs; Figure 2 middle). Exceeding the second
threshold (>5 APs), Ca2+ signaling translates into activation of the gland jasmonic acid (JA) clock that controls stomach
formation together with the expression of hydrolases (prey processing) and transporters (nutrient absorption).

226 Trends in Plant Science, March 2018, Vol. 23, No. 3

gland transcription and the secretion of digestive enzymes can be initiated by JA alone [7].
Together, these studies demonstrate that Dionaea possesses a mechanism for counting that
resembles the biological precursor systems for elementary arithmetic which exist in many
animal species. Unlike animals, where this has been shown to be rooted in special neuronal
networks [44], counting in these carnivorous plants is probably performed by cascades of
second messenger signals and phosphorylation/dephosphorylation cycles.

Priming and Decision-Making

The very first stimuli elicited by an insect appear to prime the flytrap for subsequent decisions
[4]. The decision to initiate prey processing requires JA biosynthesis and signaling. It has
been shown that there is crosstalk, for example between JA and the water-stress hormone
abscisic acid (ABA), within plant signaling pathways [52–54]. When Dionaea is prestimulated
by ABA, trigger hair bending still elicits APs but the trap does not close on ‘two’ [7]. Trigger
hairs need to be bent at least three or even more times to elicit closure of the capture organ.
The same behavior is observed under drought conditions, when ABA levels are elevated. The
reduced touch sensitivity of Dionaea, caused by ABA, suggests that the carnivorous plant
balances the gain of capturing a prey versus the water investment needed to make use of the
expected animal-derived nutrient load. Thus, under water limitation, ABA alters the decision,
based on the count, about initiating the hunting cycle [11,47,55]. How can this be
accomplished?

Phosphorylation of proteins acts as an on–off switch in numerous physiological reactions. In the

context of the flytrap, one may therefore ask the question of whether the extent of protein
phosphorylation correlates with the count, memory, or decision-making – how does phos-
phorylation in response to the first AP or an AP series change the decision to attach importance
to passing on electrical signals?’ In this way, future phosphoproteomics studies could provide
insights into the ‘counting’ networks.

Sense of Defense
When Bemm et al. [4] compared the transcription profiles of resting and insect-stimulated
flytraps they found a pattern of differential gene expression that most closely resembles that
seen in arabidopsis after wounding or infestation by herbivores. This molecular feature sug-
gests that carnivory evolved from defense strategies against herbivores. Rewiring herbivore
defense, carnivorous plants have turned the tables, enabling them to eat animals.

Insects are protected from damage by a chitin-based shell which the flytrap must crack to get
the meat. In addition to constituting the insect coat, chitin, a nitrogen-rich N-acetyl-D-glucos-
amine polymer, is a major component of fungal cell walls and has been recognized as a general
elicitor of plant defense responses for many years [56,57]. In arabidopsis, a chitin-inducible
complex formed by the lysine motif (LysM) chitin receptor kinases AtLYK5 and AtCERK1 serves
as the bona fide chitin receptor [58]. Chitinase expression is induced upon fungal infection.
These chitin-degrading enzymes accumulate at the site of invasion. This situation is similar to
the chitin response of the flytrap (see below).

Searching for members of the Venus flytrap sensory system, more than 200 receptor-like
kinases (LRKs) were identified [4]. A Dionaea LysM-type, CERK1/LYK5-like kinase pair was
significantly upregulated following gland insect stimulation, suggesting that the glands might be
able to assess the chitin chemistry of the prey during progressive digestion, and adjust their
secretion accordingly. Nevertheless, how does the carnivorous plant decide about food and
foe when the chitin-type signals and receptors are structurally so similar?

Trends in Plant Science, March 2018, Vol. 23, No. 3 227

Chemosensing – Tasting the Meal
In response to insect attack, non-carnivorous plants do not experience a sharp AP such as that
generated by the flytrap. Instead, they generate a traveling slow electrical wave that translates
into JA biosynthesis [12,59]. Activation of the JA signaling pathway leads to the synthesis of
substances that are toxic to herbivores, and these render the plant meal indigestible for the
foraging insects.

When Darwin exposed open flytraps to all types of nutrient sources ranging from burger pieces
to nitrogen-rich chemicals [1], he realized that the capture organ slowly closed and started
secretion. From this phenomenon he concluded that, in addition to its touch sensor, Dionaea
operates a chemosensing system that assesses the quality of the food provided by the prey.

Is the flytrap able to sense and process chitin? A major fraction of the lytic enzyme cocktail
secreted into the green stomach consists of chitinases [60]. To test whether Dionaea is able to
sense and respond to chitin, Bemm et al. [4] studied the stimulus-induced gland expression of
the major chitinase CHIB [61]. Traps were first stimulated mechanically and CHIB expression
was monitored. Following stimulation onset, transcriptional activation of hydrolase genes was
observed within 1 hour. After several hours, a second touch stimulus induced CHIB expression
almost 400-fold. If the second stimulus was chitin, CHIB expression was boosted up to 2000-
fold. This indicates that, depending on the history of the mechanostimulus, a subsequent
chemical stimulus such as chitin acts synergistically to adjust the secretion process to a given
prevailing situation. Therefore, Dionaea not only memorizes incoming signals but also inte-
grates them constantly to perfectly gear prey digestion and nutrient uptake.

Glands Process the Prey

Morphological studies have revealed that each gland is composed of 46 cells arranged in three
layers (Figure 3) [14,62,63]. In resting glands the innermost stalk cells are tightly packed with
oleosomes, suggesting that a triacylglycerol reservoir might represent the primary currency for
energy consumption processes. In line with this observation is the finding that transcripts
involved in triacylglycerol (TAG) breakdown, including peroxisomal b-oxidation, are highly
expressed in gland tissue [4]. The pronounced appearance of plasma-membrane invaginations
at the basal end of upper-layer cells and throughout the body of middle-layer cells is indicative
of an increased interface capacity for nutrient recognition and transport (see below). The
existence of an expanded rough endoplasmic reticulum together with pronounced transcrip-
tional activity in glands points towards a highly dynamic protein biosynthesis and translocation
machinery.

Both secretion stimulated naturally upon insect capture as well as secretion induced experi-
mentally by JA result in gross ultrastructural changes within gland cells. Middle-layer cells form
a largely invaginated plasma membrane that extends entirely over stimulated cells. As a result,
glands increase their surface-to-volume ratio [7]. Analysis of enriched transcripts in active trap
tissue shows that trap stimulation induces secretion-associated transcriptional activity [4].
Among the differentially expressed genes, transcripts exhibiting a secretion-related export
signal were subject to massive induction. These included transcripts encoding secreted
proteins with hydrolyzing activity, such as proteases, phosphatases, and chitinases. Depend-
ing on the history and frequency of mechanostimulation, digestive enzyme production and
secretion were found to be adjusted to ongoing prey decomposition.

The presence of vesicles in the cytosol of resting glands, as well as the observation of numerous
membrane invaginations after stimulation [10], point towards exocytotic secretion of

228 Trends in Plant Science, March 2018, Vol. 23, No. 3

components required for the digestion of prey. In animal cells techniques have been developed
to study exocytotic secretion at the single-cell level, including the detection of individual
secretory events [64,65]. Ion-selective electrodes [66] and carbon fiber amperometry [10]
have recently been applied to study secretion from Dionaea glands [10] (Box 1 [629_TD$IF]). Amperometric
spikes result from the oxidation of substances released from single vesicles – most likely
glutathione (GSH). Unlike similar studies in animal cells, where such spikes have durations of
milliseconds, spikes recorded from the surface of Dionaea glands had half-widths in the range
of several seconds. This difference is most likely due to the fact that released substances must
cross the gland cell wall. Secretory events started to appear about 12 h after stimulation
(regardless of whether the trigger was mechanical or of the jasmonate type), and their
frequency stayed elevated for about 2 days. This timecourse resembles the appearance of
transcripts of the vacuolar-type proton pump, as well as for enzymes involved in GSH
biosynthesis. Time-resolved nuclear magnetic resonance studies show that, within this
time-period, the sealed trap is filled up with digestive fluid that covers the prey entirely [10].

Mining the Nutrient Stock of the Animal

While digesting the victim, metabolites and minerals containing the essential plant nutrients
nitrogen, phosphorus, and sulfur are released. Analysis of RNAs encoding solute transporters,
with particular focus on those upregulated by insect feeding, identified transcripts encoding

Box 1. Monitoring Exocytosis in Dionaea

Constitutive exocytosis in plants controls cell growth and morphogenesis as well as the turnover and distribution of
membrane proteins. Triggered exocytosis has been found in association with herbivory, wounding, and the hormone
jasmonate. Exocytotic vesicles are generated at the endoplasmic reticulum, pass through the Golgi apparatus, and
deliver phospholipids, together with membrane proteins, to the plasma membrane, while their contents are released
into the apoplast.

Monitoring of single secretory events by electrophysiological methods has allowed the triggered secretion processes to
be observed in detail.

Vibrating ion-selective microelectrodes provide a method for the quantification of the vesicle proton load upon fusion
with the plasma membrane. In brief, pH-selective electrodes move between two positions: close to and distant from the
cell surface. The voltage characteristics recorded at the two positions are converted into concentration parameters.
Finally, the net H+ fluxes can then be calculated from the measured voltage gradient (Figure I, blue trace, upper left).

Amperometry electrochemically detects vesicle fusion events by monitoring changes in the redox potential at the cell
surface. A polarized carbon fiber-based electrode is kept at a fixed voltage and placed in close proximity to the cell
surface. Upon vesicular release of a redox-active compound a characteristic current signal is measured at the electrode
that results from oxidation or reduction of the target molecule. Thereby, the ‘quantal’ release of secreted molecules is
resolved with high temporal and spatial precision (Figure I, red trace, upper right).

A combination of different techniques in the non-model plant Dionaea were applied successfully to resolve the
timecourse of stimulus-induced exocytosis as well as the molecular identity of the cargo secreted.

Early vesicles (Figure I, blue): within 2 minutes after stimulation fluid phase secretion via H+- and Cl -containing vesicles
can be recorded (Figure I, blue trace, upper left). According to ion-selective electrode measurements, the exocytosis of
acidic vesicles reaches a peak several hours after stimulation onset. Transcriptomic analyses during the early time-
course of secretion led to the identification of ion transporters underlying vesicle acidification: P- and V-type proton-
pump ATPases together with a CLC-type chloride/proton antiporter drive HCl loading.

Late vesicles (Figure I, red): ongoing mechanostimulation by the struggling prey finally results in enhanced expression of
lytic enzymes and digestive compounds. Among these compounds, GSH is responsible for controlling the redox state
of the digestive fluid. Transcriptomic profiling identified GSH transporters and hydrolases. These late vesicles containing
the digestive cargo were detected amperometrically only 12 h after stimulation, and vesicle release lasted for several
days. Finally, protein composition of the digestive fluid was determined in a proteomic approach.

Trends in Plant Science, March 2018, Vol. 23, No. 3 229

 Vibrang electrode Amperometry
(minutes to hours) (hours to days)

H+ flux

2 pA
50 min 10 min
12 h

GSH
HCI Hydrolases
HCI
ADP + Pi ATP
ADP + Pi
ATP KT1 +
K
H+ +
H+ H − Hydrolases
Cl GSH
Cl− − ATP HCI OPT4
Cl
AHA10 ADP + Pi
VHA GSH
H+
H + H+
Cl−

CLC H
+

Golgi

Figure I. Exocytosis-Coupled Secretion of Dionaea Glands.

transport proteins for amino acids, sulfate, and phosphorus, as well as those for minerals (see
below). We detail here the uptake by glandular transporters for ammonium, potassium, and
sodium.

Ammonium
The Venus flytrap uses nitrogen from prey-derived amino acids to synthesize new proteins,
while the carbon backbone fuels respiration [67]. When insect powder was incubated with the
digestive enzyme mixture of the Venus flytrap ‘green stomach’, both amino acids and NH4+[628_TD$IF]
were released. Gland cells of the digesting Dionaea stomach bring an ammonium uptake
system into action. In line with increased de novo biosynthesis of an ammonium transporter,
DmAMT1, high transcript levels for this protein were found, especially in stimulated glands [68].
Functional characterization of DmAMT1 in Xenopus oocytes revealed that DmAMT1 exhibits all
the hallmark biophysical properties of a NH4+-selective uptake channel.

Potassium and Sodium

Metal ions including potassium and sodium are associated with a meat diet. Compared to
untriggered glands, stimulated glands could be depolarized more strongly by application of
external K+ and Na+ [22,69]. This indicates that, upon stimulation, sodium and potassium
transporters are incorporated into the plasma membrane of gland cells. In searching the
Dionaea reference transcriptome for sodium channels, no animal-type sodium channel

230 Trends in Plant Science, March 2018, Vol. 23, No. 3

sequences were found. However, DmHKT1, an ortholog of an archetypical cation channel [70],
was identified. In addition, the K+ channel DKT1 together with the DmHAK5-type potassium
transporter were found to be highly expressed [22]. Moreover, jasmonates dramatically
induced DmAMT1, DmHKT1, and DmHAK5 expression in the trap, and particularly in secreting
glands [22,35,69]. Last but not least, simulating a struggling insect by eliciting 2–60 APs
manually led to an increase of trap transporter expression in a count-dependent manner. Thus,
mechanical stimulation is the key factor controlling transporter expression and cation-induced
depolarization. The finding that JA biosynthesis follows mechanosensing suggests that this
phytohormone not only controls prey hydrolysis [8,71,72] but also the uptake of released
nutrients.

Concluding Remarks and Future Directions

How To Build a Mechanosensitive Organ?
Trigger hairs enable the flytrap to count prey contacts (APs) as a basis for making decisions.
How much force does an insect need to bend the trigger hair enough to elicit an AP?
Understanding the biomechanics and haptoelectrics of the process will require knowledge
about the molecular mechanisms that control the trigger hair. As a first step towards under-
standing the molecular inventory of the Dionaea mechanosensor, the mechanosensory cells
might be isolated and the profile of gene expression determined. Channels may thereby be
identified as likely candidates for the receptor potential and AP generation. Among them,
mechanosensitive and Ca2+-permeable channels together with voltage-activated channels
may be identified.

The Evolution of Counting and Carnivory

Mechanosensitive trigger hairs and snap-traps are not only found in Dionaea but also in its
aquatic little sister – the waterwheel Aldrovanda [73]. Both plants share a common ancestor
within the genus Drosera that also includes carnivorous plants. Members of this genus grow
leaves of various shapes and a multitude of glandular emergences [74,75]. Phylogenetic
studies suggest that trigger hairs have their root in mechanosensitive tentacles seen in some
Drosera family members [75]. Thus, it would be interesting to compare the genetic makeup of
trigger hairs with that of primitive and advanced Drosera tentacles in the future.

From detailed transcriptome analysis it was evident that the Venus flytrap is a green plant [4].
Electrical excitability does not come from voltage-dependent Na+ and Ca2+ channels. Likewise,
the fast movement is not generated by a green muscle. Thus, the carnivorous plant has not
gained its hunting skills by horizontal gene transfer from prey animals, but from rewiring
elements of defense pathways and leaf development programs that are even shared with
cabbage.

The most advanced trap type, the snap-trap, is likely to have evolved only once in plants,
namely in the last common ancestor of Aldrovanda and Dionaea [35,73–76]. Capturing larger
prey appears to be the main selective driving force for the evolution of snap-traps and for the
elongated leaves and fast tentacles in the genus Drosera [74]. In even more advanced Drosera,
the leaf blades wrap around the prey.

To trace the interfamily evolution of traps and the emergence of counting, the expansion and
function of genes/families in Dionaea and Aldrovanda will need to be compared to those of
primitive and more advanced Drosera species. Correlations between the emergence of
these genes with the ability to count may point to the roots of this amazing skill of a green
plant.

Trends in Plant Science, March 2018, Vol. 23, No. 3 231

Learning from Mutants of Non-Model Organisms Outstanding Questions
Having generated genomic and/or detailed transcriptomic data for Dionaea, we are still facing What is the molecular basis of the car-
concerns because non-model organisms are ‘not offering numerous mutants’ and currently have nivorous lifestyle of Dionaea?

‘no established transformation’ protocol [77,78]. Spontaneous mutations occur frequently in

How can we understand a plant that
Dionaea muscipula, and a large number of different phenotypes are currently available. The ‘hunts animals’, if these skills do not
research community has optimized the in vitro organogenesis of shoots from petiole explants, thus exist in the model plant arabidopsis?
providing an ideal basis for in vitro mutagenesis and probably also for an Agrobacterium tume-
faciens-mediated transformation system. Such a mutagenesis approach would result in random Dionaea is an excitable plant that fires
APs when in contact with prey. How
mutants and enable the identification of novel and so far unknown gene functions.
are the APs generated, and what type
of electrical network is involved?
How To Confirm Candidate Genes with a Function in Generating the AP?
While a collection of Dionaea mutant and transgenic lines remains to be established [619_TD$IF](see How is Dionaea able to learn about the
Outstanding Questions), what can we learn from a model system? For example, the touch- size of the meal by counting the num-
ber of fired APs?
sensitive plant Mimosa pudica has long attracted the interest of researchers. Seismonastic
closing movements of its leaflets and leaves is preceded by mechanically induced APs. While
The closed trap encaging the prey
genome sequencing and transcriptome analysis is still ongoing, an efficient genetic transfor- turns into a ‘green stomach’. How
mation method for M. pudica has already been developed [79]. Stable integration of genetically does the series of APs address the
encoded Ca2+[630_TD$IF]-and voltage-dependent sensors [80,81] could be tested for effects on AP endocrine system? How is the prey
digested, and how are the animal-
generation and the elicitation of Ca2+ waves [26]. Alternatively, directed mutagenesis of AP
derived nutrients taken up?
channel candidates via CRISPR/Cas9 [82,83] could be performed. This way, the interconnec-
tion between electrical and Ca2+ waves may be studied [12,26]. The Mimosa AP is slower and
of different shape than that of Dionaea. It will thus be interesting to see which of the flytrap ion
channels, when incorporated into Mimosa, will affect AP speed and shape as well as (or not)
altering touch-induced leaf movement.

Any proper channels identified in the touch-sensing expert plants Dionaea and Mimosa could
be introduced into arabidopsis wild-type and mutants. This would potentially transform touch/
wound-induced electrical signals into real APs that might also affect stomatal closure, for
example.

Concluding Remarks
Together with a recent editorial [84], we would like to argue in favor of analyzing the extraordi-
nary skills of ‘unusual creatures’. When Darwin explored the development of species he did not
concentrate on a somewhat limited set of ‘model’ organisms. Instead, he chose his objects of
study on the basis of their suitability with respect to a particular question. When working with the
mechanosensory and signaling networks of the Venus flytrap – including its ability to memorize
and count – he very likely would have studied this side by side with the nervous systems of
simple animals such as sea slugs [85,86]. Unquestionably, Darwin would have applied any
available molecular genetic methods for conducting functional studies on species of his current
biological interest, rather than using methods that were restricted to, or even developed only
for, model organisms.

Acknowledgments
This work was supported by the European Research Council (ERC) under the EU 7th Framework Program (FP/20010-
2015)/ERC grant agreement 250194 Carnivorom. We thank Dirk Becker, Ines Fuchs, and Sönke Scherzer for helpful
discussions.

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