Demystifying the Venus flytrap action potential.

All plants are electrically excitable, but only few are known to fire a well-defined, all-or-nothing action potential (AP). The Venus flytrap Dionaea muscipula displays APs with an extraordinarily high firing frequency and speed, enabling the capture organ of this carnivorous plant to catch small animals as fast as flies. The number of APs triggered by the prey is counted and serves as the basis for decisions within the flytrap's hunting cycle. The archetypical Dionaea AP lasts 1 s and consists of five phases: Starting from the resting state, an initial cytosolic Ca2+ transient is followed by depolarization, repolarization and a transient hyperpolarization (overshoot) before the original membrane potential is finally recovered. When the flytrap matures and becomes excitable, a distinct set of ion channels, pumps and carriers is expressed, each mastering a distinct AP phase.

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

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

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

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

Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap.

Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca2+ wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K+ channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap's prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca2+ transients, we, in mature trigger hairs firing fast Ca2+ signals and APs, found OSCA1.7 and GLR3.6 type Ca2+ channels and ACA2/10 Ca2+ pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca2+ and electrical event. Given that anesthetics affect K+ channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca2+ and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca2+ activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.

The Venus flytrap trigger hair-specific potassium channel KDM1 can reestablish the K+ gradient required for hapto-electric signaling.

The carnivorous plant Dionaea muscipula harbors multicellular trigger hairs designed to sense mechanical stimuli upon contact with animal prey. At the base of the trigger hair, mechanosensation is transduced into an all-or-nothing action potential (AP) that spreads all over the trap, ultimately leading to trap closure and prey capture. To reveal the molecular basis for the unique functional repertoire of this mechanoresponsive plant structure, we determined the transcriptome of D. muscipula's trigger hair. Among the genes that were found to be highly specific to the trigger hair, the Shaker-type channel KDM1 was electrophysiologically characterized as a hyperpolarization- and acid-activated K+-selective channel, thus allowing the reuptake of K+ ions into the trigger hair's sensory cells during the hyperpolarization phase of the AP. During trap development, the increased electrical excitability of the trigger hair is associated with the transcriptional induction of KDM1. Conversely, when KDM1 is blocked by Cs+ in adult traps, the initiation of APs in response to trigger hair deflection is reduced, and trap closure is suppressed. KDM1 thus plays a dominant role in K+ homeostasis in the context of AP and turgor formation underlying the mechanosensation of trigger hair cells and thus D. muscipula's hapto-electric signaling.

Genomes of the Venus Flytrap and Close Relatives Unveil the Roots of Plant Carnivory.

Most plants grow and develop by taking up nutrients from the soil while continuously under threat from foraging animals. Carnivorous plants have turned the tables by capturing and consuming nutrient-rich animal prey, enabling them to thrive in nutrient-poor soil. To better understand the evolution of botanical carnivory, we compared the draft genome of the Venus flytrap (Dionaea muscipula) with that of its aquatic sister, the waterwheel plant Aldrovanda vesiculosa, and the sundew Drosera spatulata. We identified an early whole-genome duplication in the family as source for carnivory-associated genes. Recruitment of genes to the trap from the root especially was a major mechanism in the evolution of carnivory, supported by family-specific duplications. Still, these genomes belong to the gene poorest land plants sequenced thus far, suggesting reduction of selective pressure on different processes, including non-carnivorous nutrient acquisition. Our results show how non-carnivorous plants evolved into the most skillful green hunters on the planet.

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

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

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

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

Venus flytrap carnivorous lifestyle builds on herbivore defense strategies.

Although the concept of botanical carnivory has been known since Darwin's time, the molecular mechanisms that allow animal feeding remain unknown, primarily due to a complete lack of genomic information. Here, we show that the transcriptomic landscape of the Dionaea trap is dramatically shifted toward signal transduction and nutrient transport upon insect feeding, with touch hormone signaling and protein secretion prevailing. At the same time, a massive induction of general defense responses is accompanied by the repression of cell death-related genes/processes. We hypothesize that the carnivory syndrome of Dionaea evolved by exaptation of ancient defense pathways, replacing cell death with nutrient acquisition.

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.

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

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

Stomatal guard cells co-opted an ancient ABA-dependent desiccation survival system to regulate stomatal closure.

During the transition from water to land, plants had to cope with the loss of water through transpiration, the inevitable result of photosynthetic CO2 fixation on land [1, 2]. Control of transpiration became possible through the development of a new cell type: guard cells, which form stomata. In vascular plants, stomatal regulation is mediated by the stress hormone ABA, which triggers the opening of the SnR kinase OST1-activated anion channel SLAC1 [3, 4]. To understand the evolution of this regulatory circuit, we cloned both ABA-signaling elements, SLAC1 and OST1, from a charophyte alga, a liverwort, and a moss, and functionally analyzed the channel-kinase interactions. We were able to show that the emergence of stomata in the last common ancestor of mosses and vascular plants coincided with the origin of SLAC1-type channels capable of using the ancient ABA drought signaling kinase OST1 for regulation of stomatal closure.

Integration of trap- and root-derived nitrogen nutrition of carnivorous Dionaea muscipula.

Carnivorous Dionaea muscipula operates active snap traps for nutrient acquisition from prey; so what is the role of D. muscipula's reduced root system? We studied the capacity for nitrogen (N) acquisition via traps, and its effect on plant allometry; the capacity of roots to absorb NO3(-), NH4(+) and glutamine from the soil solution; and the fate and interaction of foliar- and root-acquired N. Feeding D. muscipula snap traps with insects had little effect on the root : shoot ratio, but promoted petiole relative to trap growth. Large amounts of NH4(+) and glutamine were absorbed upon root feeding. The high capacity for root N uptake was maintained upon feeding traps with glutamine. High root acquisition of NH4(+) was mediated by 2.5-fold higher expression of the NH4(+) transporter DmAMT1 in the roots compared with the traps. Electrophysiological studies confirmed a high constitutive capacity for NH4(+) uptake by roots. Glutamine feeding of traps inhibited the influx of (15)N from root-absorbed (15)N/(13)C-glutamine into these traps, but not that of (13)C. Apparently, fed traps turned into carbon sinks that even acquired organic carbon from roots. N acquisition at the whole-plant level is fundamentally different in D. muscipula compared with noncarnivorous species, where foliar N influx down-regulates N uptake by roots.

Influence of the sevoflurane concentration on the occurrence of epileptiform EEG patterns.

OBJECTIVES AND AIM: This study was performed to analyse the effects of different sevoflurane concentrations on the incidence of epileptiform EEG activity during induction of anaesthesia in children in the clinical routine. BACKGROUND: It was suggested in the literature to use sevoflurane concentrations lower than 8% to avoid epileptiform activity during induction of anaesthesia in children. METHODS: 100 children (age: 4.6±3.0 years, ASA I-III, premedication with midazolam) were anaesthetized with 8% sevoflurane for 3 min or 6% sevoflurane for 5 min in 100% O2 via face mask followed by 4% sevoflurane until propofol and remifentanil were given for intubation. EEGs were recorded continuously and were analysed visually with regard to epileptiform EEG patterns. RESULTS: From start of sevoflurane until propofol/remifentanil administration, 38 patients (76%) with 8% sevoflurane had epileptiform EEG patterns compared to 26 patients (52%) with 6% (p = 0.0106). Epileptiform potentials tended to appear later in the course of the induction with 6% than with 8%. Up to an endtidal concentration of 6% sevoflurane, the number of children with epileptiform potentials was similar in both groups (p = 0.3708). The cumulative number of children with epileptiform activity increased with increasing endtidal sevoflurane concentrations. The time from start of sevoflurane until loss of consciousness was similar in patients with 8% and 6% sevoflurane (42.2±17.5 s vs. 44.9 s ±14.0 s; p = 0.4073). An EEG stage of deep anaesthesia with continuous delta waves <2.0 Hz appeared significantly earlier in the 8% than in the 6% group (64.0±22.2 s vs. 77.9±20.0 s, p = 0.0022). CONCLUSION: The own analysis and data from the literature show that lower endtidal concentrations of sevoflurane and shorter administration times can be used to reduce epileptiform activity during induction of sevoflurane anaesthesia in children.

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

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

Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.

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

The Dionaea muscipula ammonium channel DmAMT1 provides NH4⁺ uptake associated with Venus flytrap's prey digestion.

BACKGROUND: Ammonium transporter (AMT/MEP/Rh) superfamily members mediate ammonium uptake and retrieval. This pivotal transport system is conserved among all living organisms. For plants, nitrogen represents a macronutrient available in the soil as ammonium, nitrate, and organic nitrogen compounds. Plants living on extremely nutrient-poor soils have developed a number of adaptation mechanisms, including a carnivorous lifestyle. This study addresses the molecular nature, function, and regulation of prey-derived ammonium uptake in the Venus flytrap, Dionaea muscipula, one of the fastest active carnivores. RESULTS: The Dionaea muscipula ammonium transporter DmAMT1 was localized in gland complexes where its expression was upregulated upon secretion. These clusters of cells decorating the inner trap surface are engaged in (1) secretion of an acidic digestive enzyme cocktail and (2) uptake of prey-derived nutrients. Voltage clamp of Xenopus oocytes expressing DmAMT1 and membrane potential recordings with DmAMT1-expressing Dionaea glands were used to monitor and compare electrophysiological properties of DmAMT1 in vitro and in planta. DmAMT1 exhibited the hallmark biophysical properties of a NH4(+)-selective channel. At depolarized membrane potentials (Vm = 0), the Km (3.2 ± 0.3 mM) indicated a low affinity of DmAMT1 for ammonium that increased systematically with negative going voltages. Upon hyperpolarization to, e.g., -200 mV, a Km of 0.14 ± 0.015 mM documents the voltage-dependent shift of DmAMT1 into a NH4(+) transport system of high affinity. CONCLUSIONS: We suggest that regulation of glandular DmAMT1 and membrane potential readjustments of the endocrine cells provide for effective adaptation to varying, prey-derived ammonium sources.

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues.

BACKGROUND: Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. RESULTS: Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. CONCLUSION: Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.