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The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na\(^+\)- and K\(^+\)-permeable mutants function as ion channels rather than K\(^+\) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na\(^+\)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.
The Venus Flytrap Dionaea muscipula Counts Prey-Induced Action Potentials to Induce Sodium Uptake
(2016)
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.
Plants are able to sense mechanical forces in order to defend themselves against predators,
for instance by synthesizing repellent compounds. Very few plants evolved extremely sensitive
tactile abilities that allow them to perceive, interpret and respond by rapid movement in the
milliseconds range. One such rarity is the charismatic Venus flytrap (Dionaea muscipula) - a
carnivorous plant which relies on its spectacular active trapping strategy to catch its prey. The
snapping traps are equipped with touch-specialised trigger hairs, that upon bending elicit an
action potential (AP). This electrical signal originates within the trigger hairs’ mechanosensory
cells and further propagates throughout the whole trap, alerting the plant of potential prey.
Two APs triggered within thirty seconds will set off the trap and more than five APs will
initiate the green stomach formation for prey decomposition and nutrient uptake. Neither
the molecular components of the plant’s AP nor the Venus flytrap’s fast closure mechanism
have been fully elucidated yet. Therefore, the general objective of this study is to expound
on the molecular basis of touch perception: from AP initiation to trap closure and finally to
stomach formation.
The typical electrical signal in plants lasts for minutes and its shape is determined by the
intensity of the mechanical force applied. In contrast, the Venus flytrap’s one-second AP is of
all-or-nothing type, similar in shape to the animal AP. In order to gain more insight into the
molecular components that give rise to the Venus flytrap’s emblematic AP, the transcriptomic
landscape of its unique mechanotransducer - the trigger hair – was compared to the rest
of the non-specialised tissues and organs. Additionally, the transcriptome of the electrically
excitable fully-developed adult trap was compared to non-excitable juvenile traps that are
unable to produce sharp APs. Together, the two strategies helped with the identification of
electrogenic channels and pumps for each step of the AP as follows: (1) the most specific to
the trigger hair was the mechanosensitive channel DmMSL10, making up the best candidate for
the initial AP depolarization phase, (2) the K+ outward rectifier DmSKOR could be responsible
for repolarisation, (3) further, the proton pump DmAHA4, might kick in during repolarisation
and go on with hyperpolarisation and (4) the hyperpolarization- and acid-activated K+ inward
rectifier KDM1 might contribute to the re-establishment of electrochemical gradient and
the resting potential. Responsible for the AP-associated Ca2+ wave and electrical signal
propagation, the glutamate-like receptor DmGLR3.6 was also enriched in the trigger hairs.
Together, these findings suggest that the reuse of genes involved in electrical signalling in
ordinary plants can give rise to the Venus flytrap’s trademark AP.
The Venus flytrap has been cultivated ever since its discovery, generating more than one
hundred cultivars over the years. Among them, indistinguishable from a normal Venus flytrap
at first sight, the ’ERROR’ cultivar exhibits a peculiar behaviour: it is unable to snap its traps
upon two APs. Nevertheless, it is still able to elicit normal APs. To get a better understanding
of the key molecular mechanisms and pathways that are essential for a successful trap closure,
the ’ERROR’ mutant was compared to the functional wild type.
Timelapse photography led to the observation that the ’ERROR’ mutants were able to leisurely
half close their traps when repeated mechanostimulation was applied (10 minutes after 20
APs, 0.03 Hz). As a result of touch or wounding in non-carnivorous plants, jasmonic acid
(JA) is synthesized, alerting the plants of potential predators. Curiously, the JA levels were reduced upon mechanostimulation and completely impaired upon wounding in the ’ERROR’
mutant. In search of genes accountable for the ’ERROR’ mutant’s defects, the transcriptomes
of the two phenotypes were compared before and after mechanostimulation (1h after 10
APs, 0.01 Hz). The overall dampened response of the mutant compared to the wild type,
was reflected at transcriptomic level as well. Only about 50% of wild type’s upregulated
genes after touch stimulation were differentially expressed in ’ERROR’ and they manifested
only half of the wild type’s expression amplitude. Among unresponsive functional categories
of genes in ’ERROR’ phenotype, there were: cell wall integrity surveilling system, auxin
biosynthesis and stress-related transcription factors from the ethylene-responsive AP2/ERF and
C2H2-ZF families. Deregulated Ca2+-decoding as well as redox-related elements together with
JA-pathway components might also contribute to the malfunctioning of the ’ERROR’ mutant. As
the mutant does not undergo full stomach formation after mechanical treatment, these missing
processes represent key milestones that might mediate growth-defence trade-offs under JA
signalling. This confirms the idea that carnivory has evolved by recycling the already available
molecular machineries of the ubiquitous plant immune system.
To better understand the mutant’s defect in the trap snapping mechanism, the ground states
(unstimulated traps) of the two phenotypes were compared. In this case, many cell wall-related
genes (e.g. expansins) were downregulated in the ’ERROR’ mutant. For the first time, these
data point to the importance of a special cell wall architecture of the trap, that might confer
the mechanical properties needed for a functional buckling system - which amplifies the speed
of the trap closure.
This study provides candidate channels for each of the AP phases that give rise to and shape
the sharp Venus flytrap-specific AP. It further underlines the possible contribution of the cell
wall architecture to the metastable ready-to-snap configuration of the trap before stimulation
- which might be crucial for the buckling-dependent snapping. And finally, it highlights
molecular milestones linked to defence responses that ensure trap morphing into a green
stomach after mechanostimulation. Altogether, these processes prove to be interdependent
and essential for a successful carnivorous lifestyle.
Die Venusfliegenfalle, Dionaea muscipula, weckte aufgrund ihrer karnivoren Lebensweise schon sehr früh das Interesse vieler Wissenschaftler. Für karnivore Pflanzen, die auf Nährstoff-armen Böden wachsen, spielen Insekten als Beute und somit als Nährstofflieferant eine entscheidende Rolle. So können die Pflanzen durch die Verdauung der Beute mit wichtigen Makro- und Mikronährstoffen, wie Stickstoff, Phosphat, Kalium oder Natrium versorgt werden. Aus diesem Grund sollte im Rahmen meiner Arbeit ein besonderes Augenmerk auf die molekularen Mechanismen der Kationenaufnahme während der Nährstoffresorption gerichtet werden. Insbesondere die aus dem Insekt stammenden Nährstoffe Kalium und Natrium waren dabei von großem Interesse.
Im Allgemeinen sind Kaliumionen für Pflanzen eine essentielle anorganische Substanz und von großer physiologischer Bedeutung für die Entwicklung, den Metabolismus, die Osmoregulation, das Membranpotential und viele zelluläre Prozesse. Analysen der Kaliumaufnahme an Wurzeln von Modellpflanzen wie Arabidopsis thaliana und Reis zeigten, dass die Aufnahme von K+ ein Zusammenspiel von hoch-affinen K+-Transportern der HAK5-Familie und nieder-affinen Kaliumkanälen (AKT1/AtKC1) erfordert, die in ein komplexes
(De-)Phosphorylierungsnetzwerk eingebunden sind. In der vorliegenden Arbeit war es mir möglich das Netzwerk zur Kaliumaufnahme in den Drüsen der Venusfliegenfalle zu entschlüsseln. Es konnten Orthologe zum Kaliumtransporter HAK5 aus Arabidopsis (DmHAK5) und zum Kaliumkanal AKT1 (DmKT1) identifiziert und im heterologen Expressionssystem der Xenopus laevis Oozyten elektrophysiologisch charakterisiert werden. Dabei zeigte sich, das DmKT1 durch einen Ca2+-Sensor/Kinase-Komplex aus der CBL/CIPK-Familie phosphoryliert und somit aktiviert wird. Phylogenetische Analysen von DmKT1 bestätigten die Eingruppierung dieses Kaliumkanals in die Gruppe der pflanzlichen Shaker-Kaliumkanäle des AKT1-Typs. Die Transporteigenschaften zeigten zudem, dass DmKT1 bei hyperpolarisierenden Membranpotentialen aktiviert wird und einen K+-selektiven Einwärtsstrom vermittelt. In Oozyten konnte eine Kaliumaufnahme bis zu einer externen Konzentration von ≥1 mM beobachtet werden. DmKT1 repräsentiert also einen Kaliumkanal mit einer hohen Transportkapazität, der die nieder-affine Kaliumaufnahme in die Drüsenzellen der Venusfliegenfalle vermitteln kann.
Unterhalb einer externen Kaliumkonzentration von 1 mM würde der anliegende elektrochemische Kaliumgradient einen Kaliumausstrom und somit einen Verlust von Kalium favorisieren. Hoch-affine K+/H+-Symporter können durch die Ausnutzung des Protonengradienten eine Kaliumaufnahme im mikromolaren Bereich gewährleisten. In Wurzelhaaren von Arabidopsis vermittelt der Transporter AtHAK5 die Kaliumaufnahme unter Kaliummangelbedingungen. DmHAK5, ein Ortholog zu AtHAK5, ist in Dionaea Drüsen exprimiert und konnte zum ersten Mal im heterologen Expressionssystem der Xenopus Oozyten im Detail charakterisiert werden. Interessanterweise zeigte sich, dass DmHAK5 wie der K+-Kanal DmKT1 durch denselben CBL/CIPK-Komplex posttranslational reguliert und aktiviert wird. Die Transporteigenschaften von DmHAK5 wiesen auf einen Transporter mit einer breiten Substratspezifität hin, sodass sich DmHAK5 neben Kalium auch für Ammonium permeabel zeigte. Affinitätsuntersuchungen von DmHAK5 zu seinem Substrat Kalium klassifizierten das Protein als einen hoch-affinen Kaliumtransporter, der im Symport mit Protonen die Kaliumaufnahme im mikromolaren Konzentrationsbereich vermitteln kann.
Das Kaliumtransportmodul besteht also aus dem K+-selektiven Kanal DmKT1 und dem
K+/H+-Symporter DmHAK5, die die hoch- und nieder-affine Kaliumaufnahme in den Drüsenzellen während der Beuteverdauung in Dionaea muscipula Fallen ermöglichen. Beide Transportmodule werden Kalzium-abhängig durch die Kinase CIPK23 und den Ca2+-Sensor CBL9 auf posttranslationaler Ebene reguliert.
Zusammenfassend gelang es in dieser Arbeit Einblicke in die Kationenaufnahme während der Nährstoffresorptionsphase der Venusfliegenfalle, Dionaea muscipula, zu gewinnen. Dabei wurde klar, dass Dionaea muscipula im Laufe ihrer Evolution zu einer karnivoren Pflanze, nicht neue Transportmodule zur Nährstoffresorption aus der Beute entwickelte, sondern bekannte aus Wurzeln stammende Transportmodule umfunktionierte. Auf molekularer Ebene konnten die biophysikalischen Charakteristika der K+- und Na+-Transportproteine, sowie ihre Regulation entschlüsselt werden. Diese Erkenntnisse wurden schließlich in den Kontext des Beutefangs der Venusfliegenfalle gebracht und diskutiert.