María Escalante-Pérez

A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap

Venus flytraps leaves can catch an insect in a fraction of a second. Since the time of Charles Darwin, scientists have struggled to understand the sensory biology and biomechanics of this plant, Dionaea muscipula. Here we show that insect-capture of Dionaea traps is modulated by the phytohormone abscisic acid (ABA) and jasmonates. Water-stressed Dionaea, as well as those exposed to the drought-stress hormone ABA, are less sensitive to mechanical stimulation. In contrast, application of 12-oxo-phytodienoic acid (OPDA), a precursor of the phytohormone jasmonic acid (JA), the methyl ester of JA (Me-JA), and coronatine (COR), the molecular mimic of the isoleucine conjugate of JA (JA-Ile), triggers secretion of digestive enzymes without any preceding mechanical stimulus. Such secretion is accompanied by slow trap closure. Under physiological conditions, insect-capture is associated with Ca2+ signaling and a rise in OPDA, Apparently, jasmonates bypass hapto-electric processes associated with trap closure. However, ABA does not affect OPDA-dependent gland activity. Therefore, signals for trap movement and secretion seem to involve separate pathways. Jasmonates are systemically active because application to a single trap induces secretion and slow closure not only in the given trap but also in all others. Furthermore, formerly touch-insensitive trap sectors are converted into mechanosensitive ones. These findings demonstrate that prey-catching Dionaea combines plant-specific signaling pathways, involving OPDA and ABA with a rapidly acting trigger, which uses ion channels, action potentials, and Ca2+ signals.

The Protein Composition of the Digestive Fluid from the Venus Flytrap Sheds Light on Prey Digestion Mechanisms

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plants digestive system evolved from defense-related processes. This in-depth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plants prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition.

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.

Venus flytrap carnivorous lifestyle builds on herbivore defense strategies

Although the concept of botanical carnivory has been known since Darwins 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.

Poplar extrafloral nectaries: two types, two strategies of indirect defenses against herbivores.

Many plant species grow extrafloral nectaries and produce nectar to attract carnivore arthropods as defenders against herbivores. Two nectary types that evolved with Populus trichocarpa (Ptr) and Populus tremula × Populus tremuloides (Ptt) were studied from their ecology down to the genes and molecules. Both nectary types strongly differ in morphology, nectar composition and mode of secretion, and defense strategy. In Ptt, nectaries represent constitutive organs with continuous merocrine nectar flow, nectary appearance, nectar production, and flow. In contrast, Ptr nectaries were found to be holocrine and inducible. Neither mechanical wounding nor the application of jasmonic acid, but infestation by sucking insects, induced Ptr nectar secretion. Thus, nectaries of Ptr and Ptt seem to answer the same threat by the use of different mechanisms.

Poplar Extrafloral Nectar Is Protected against Plant and Human Pathogenic Fungus

Plants secrete nectar to attract mutualistic animals, which predominantly function as pollinators, as in the case of floral nectar, or defenders against herbivores, as in the case of extrafloral nectar (Nicolson et al., 2007). Because nectars usually represent aqueous solutions containing sugars and other nutrient metabolites (Baker and Baker, 1983), they are susceptible to infestation by microbial organisms, which can use the nectar-secreting tissues as entry sites to infect the plant. Nectar-secreting tissues thus require an efficient shield against pathogen infections. To date, our knowledge about the way that plants protect their nectar from microorganisms is rather limited. Several reports have focused on ‘defensive chemicals’, such as alkaloids and phenols, or a defense system based on proteins named nectarins (cf. Escalante—Perez and Heil, 2012). In floral nectar of ornamental tobacco, nectarins have been associated with a so-called ‘nectar redox cycle’. In this system, the production of high concentrations of reactive oxygen species (ROS) provides for microbe-free nectar. In addition to the nectarins, hydrolytic enzymes such as ribonucleases, peroxidases, chitinases, glucanases, and other pathogenesis-related (PR) proteins were discovered in both floral and extrafloral nectars (for review, see Escalante—Perez and Heil, 2012). To study the blend of antimicrobial proteins active in poplar extrafloral nectaries, we investigated the secretome and transcriptome of this organ. Thereby, we discovered a set of defense proteins that proved to be effective against a plant and human pathogenic fungus and thus could serve pharmaceutical purposes.

SLAH3‐type anion channel expressed in poplar secretory epithelia operates in calcium kinase CPK‐autonomous manner

Extrafloral nectaries secrete a sweet sugar cocktail that lures predator insects for protection from foraging herbivores. Apart from sugars and amino acids, the nectar contains the anions chloride and nitrate. Recent studies with Populus have identified a type of nectary covered by apical bipolar epidermal cells, reminiscent of the secretory brush border epithelium in animals. Border epithelia operate transepithelial anion transport, which is required for membrane potential and/or osmotic adjustment of the secretory cells. In search of anion transporters expressed in extrafloral nectaries, we identified PttSLAH3 (Populus tremula × Populus tremuloides SLAC1 Homologue3), an anion channel of the SLAC/SLAH family. When expressed in Xenopus oocytes, PttSLAH3 displayed the features of a voltage-dependent anion channel, permeable to both nitrate and chloride. In contrast to the Arabidopsis SLAC/SLAH family members, the poplar isoform PttSLAH3 is independent of phosphorylation activation by protein kinases. To understand the basis for the autonomous activity of the poplar SLAH3, we generated and expressed chimera between kinase-independent PttSLAH3 and kinase-dependent Arabidopsis AtSLAH3. We identified the N-terminal tail and, to a lesser extent, the C-terminal tail as responsible for PttSLAH3 kinase-(in)dependent action. This feature of PttSLAH3 may provide the secretory cell with a channel probably controlling long-term nectar secretion.

Mechano-Stimulation Triggers Turgor Changes Associated with Trap Closure in the Darwin Plant Dionaea muscipula

Dear Editor, The Darwin plant, also known as the Venus flytrap, Dionaea muscipula, has fascinated people since Darwin’s time. The plant lives in nutrient-poor habitats but has been able to overcome the limitations of its surroundings by evolving a carnivorous lifestyle, particularly by modifying its leaves into active traps to catch animals. When flies, ants, or other small animals touch mechano-sensitive hairs protruding from the inner surface of the bi-lobed trap, it shuts within a fraction of a second. As shown by Escalante-Perez et al. (2011), an insect bending the trigger hairs electrically excites the trap and fosters the production of the touch hormone (OPDA). Thus, visitors seem to convert formerly touch-insensitive trap sectors into mechano-sensitive ones. As insects struggle to escape, the resultant consecutive mechano-electrical stimulation increases the level of touch hormone and, as a result of hormone action, glands covering the inner epidermis of the closed trap release an acidic hydrolase cocktail (Schulze et al., 2012). Exposed to this lytic medium inside the hermetically sealed trap, the prey is digested and nitrogen-rich compounds are absorbed by the same, initially secretory, glands (Scherzer et al., 2013). Compared to the human body, therefore, the flytrap serves as mouth, stomach, and intestine all together. The fast closure of the open mouth-like Venus flytrap has been explained by two distinct mechanisms, acidic-induced cell wall loosening, or rapid loss of turgor pressure (Volkov et al., 2008). Recently, Forterre et al. (2005) suggested the importance of elastic deformations during fast trap closure. They proposed that a rapid flipping between two stable mechanical states (concave–convex) underlies trap closure. In this study, in order to monitor volume changes and displacement of trap lobes within the capture organ, which are associated with steps preceding flytrap closure, we here recorded movements of both trap lobes and sectors thereof with high-speed thermal imaging (cover photo, Figure 1 and Supplemental Movies 1 and 2). The temperature landscapes of open flytraps and visiting ants were monitored using the radiometric mode of a thermosensory infra-red camera (CMT 256 HS) equipped with a CMT sensor. We ask the question, whether ultrasensitive thermography can reveal local heat production, which might be associated with metabolic processes leading to trap closure. To induce flytrap capture through a physiological trigger, we used forceps to place ants onto capture organs whilst they were in the open state. Forcep manipulation of the ants very often resulted in wounding of the peripheral organs (e.g. antennae and legs), meaning that the ants were bleeding hemolymph. Because hemolymph transpiration cooling is associated with these wounds, the ants’ movement could be monitored as cool spots (please note color coding) as they wandered around the trap surface (cover image, Supplemental Movies 1 and 2). Ants touching a single trigger hair twice or more than one hair consecutively induced fast closure of the trap. This fast buckling of the trap’s two lobes, which has previously been observed through recordings of trap closure, has been interpreted to reflect the release of accumulated elastic energy at a distinct spot (Forterre et al., 2005). This is in parallel to our observations where no active heat production in the closing trap was obvious (Supplemental Movies 1–4). To enable reproducible monitoring of the mechanics of the capture organ, we replaced the ant with trigger hair displacements using a needle (Supplemental Movies 3 and 4). Once the mechano-sensor was touched, the open trap transiently moved relative to the camera. This change in focus/angle resulted in characteristic changes in the reflection of heat, which are merely movement artifacts (Figure 1A). Following the second mechano-stimulation, fast trap closure elicited large heat reflection signals. We quantified these signals and the kinetics of trap lobe movement after the second triggerinitiated trap lobe buckling (Figure 1). 350 ms after the second stimulus, the fast trap movement started and fast closure was completed within 500 ms. In the region where buckling originated, a transient small heat reflection (asterisks in Figure 1) preceded fast lobe movement, and we were able to consistently resolve and image this reflection from both a top (Figure 1A) and a side view (Figure 1B). These small heat reflexes point to very small changes in trap-angle, which originate from local turgor changes initiating trap closure. In the 12 experiments performed, the trapping action was associated with heat reflexes rather than heat production by the lobes. These signals evoked by high-speed high-resolution infra-red