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Comparative transcriptomics of Venus flytrap (Dionaea muscipula) across stages of prey capture and digestion.

The Venus flytrap, Dionaea muscipula, is perhaps the world's best-known botanical carnivore. The act of prey capture and digestion along with its rapidly closing, charismatic traps make this species a compelling model for studying the evolution and fundamental biology of carnivorous plants. There is a growing body of research on the genome, transcriptome, and digestome of Dionaea muscipula, but surprisingly limited information on changes in trap transcript abundance over time since feeding. Here we present the results of a comparative transcriptomics project exploring the transcriptomic changes across seven timepoints in a 72-hour time series of prey digestion and three timepoints directly comparing triggered traps with and without prey items. We document a dynamic response to prey capture including changes in abundance of transcripts with Gene Ontology (GO) annotations related to digestion and nutrient uptake. Comparisons of traps with and without prey documented 174 significantly differentially expressed genes at 1 hour after triggering and 151 genes with significantly different abundances at 24 hours. Approximately 50% of annotated protein-coding genes in Venus flytrap genome exhibit change (10041 of 21135) in transcript abundance following prey capture. Whereas peak abundance for most of these genes was observed within 3 hours, an expression cluster of 3009 genes exhibited continuously increasing abundance over the 72-hour sampling period, and transcript for these genes with GO annotation terms including both catabolism and nutrient transport may continue to accumulate beyond 72 hours.

Droseraceae↗

Digestive Secretion of Dionaea muscipula (Venus's Flytrap).

The digestive fluid of Dionaea muscipula has been studied with respect to its protein content as a function of time after entrapment of protein material and some enzymes of the secretion. Maximum secretion of enzyme occurs within the first 3 days of the digestive cycle and protein reaches its maximum at 4 days. Phosphatase, proteinase, nuclease and amylase have been observed in the secretion. The enzymes have acid pH optima and the proteinase has a molecular weight of about 40,000.

Journal Article↗

Oxidative protein modification as predigestive mechanism of the carnivorous plant Dionaea muscipula: an hypothesis based on in vitro experiments.

Aqueous leaf extracts from Dionaea muscipula contain quinones such as the naphthoquinone plumbagin that couple to different NADH-dependent diaphorases, producing superoxide and hydrogen peroxide upon autoxidation. Upon preincubation of Dionaea extracts with certain diaphorases and NADH in the presence of serumalbumin (SA), subsequent tryptic digestion of SA is facilitated. Since the secretroy glands of Droseracea contain proteases and possibly other degradative enzymes it is suggested that the presence of oxygen-activating redox cofactors in the extracts function as extracellular predigestive oxidants which render membrane-bound proteins of the prey (insects) more susceptible to proteolytic attacks.

Animals↗

Action potential and contraction of Dionaea muscipula (Venus flytrap).

Observation of the action potential and contraction of the leaf of Dionaea muscipula Ellis revealed several interesting phenomena. Two successive stimuli are generally necessary to cause contraction. The first and ineffective stimulus is associated with slow depolarization. The second stimulus has much more rapid depolarization and initiates contraction.

Action Potentials↗

Voltage-dependent K channels in protoplasts of trap-lobe cells of Dionaea muscipula.

The outward rectification of the K+ current in mesophyll cell protoplasts from trap-lobes of Dionaea muscipula was studied with the patch-clamp technique. The rectification had instantaneous and time-dependent components. Changes in [K+]i strongly affected the conductance voltage relation of the plasma membrane while changes in [K+]o had little effect on the relation. Thus, the outward rectification depends on the membrane voltage and the concentration of intracellular K+. Corresponding single-channel activities were observed both in the intact membrane (cell-attached recording) and in excised patches. The single-channel conductance was about 3.3 pS with symmetrical solutions containing 30 mM K+.

Electric Conductivity↗

Touch receptor of venous flytrap, Dionaea muscipula.

Numerous small structures (stellate trichomnes) protrude from the surface of marginal hairs, outer leaf surface, and stem of Dionaea muscipula Ellis. None are present inside the trap. Mechanical stimulation causes small action potentials and eventual closure of the trap, independently of the sensitive trigger hairs. Subthreshold stimulation of these structures appears to sensitize the trigger hairs and facilitate closure of the trap. This suiggests that these structures act as touch sensors or receptors.

In Vitro Techniques↗

Using of liquid chromatography coupled with diode array detector for determination of naphthoquinones in plants and for investigation of influence of pH of cultivation medium on content of plumbagin in Dionaea muscipula.

The interest of many investigators in naphthoquinones is due to their broad-range of biological actions from phytotoxic to fungicidal. The main aim of this work was to investigate the influence of different pH values of cultivation medium on naphthoquinone content in Dionaea muscipula. For this purpose, we optimized the simultaneous analysis of the most commonly occurring naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin) by high performance liquid chromatography coupled with diode array detector (HPLC-DAD). The most suitable chromatographic conditions were as follows: mobile phase: 0.1 mol l-1 acetic acid:methanol in ratio of 33:67 (%, v/v), flow rate: 0.75 ml min-1 and temperature: 42 degrees C. Moreover, we looked for the most suitable technique for preparation of plant samples (D. muscipula, Juglans regia, Paulownia tomentosa, Impatience glandulifera, Impatience parviflora, Drosera rotundifolia, Drosera spathulata and Drosera capensis) due to their consequent analysis by HPLC-DAD. It clearly follows from the results obtained that sonication were the most suitable technique for preparation of J. regia plants. We also checked the recoveries of the determined naphthoquinones, which were from 96 to 104%. Finally, we investigated the changes in content of plumbagin in D. muscipula plants according to different pH of cultivation medium. The content increased with increasing pH up to 5 and, then, changed gradually. The lower content of plumbagin at lower pH values was of interest to us. Therefore, we determined the content of this naphthoquinone in the cultivation medium, what has not been studied before. We discovered that the lower tissue content of plumbagin was due to secretion of this naphthoquinone into the cultivation medium.

Chromatography, High Pressure Liquid↗

Quantification of insect nitrogen utilization by the venus fly trap Dionaea muscipula catching prey with highly variable isotope signatures.

Dionaea is a highly specialized carnivorous plant species with a unique mechanism for insect capture. The leaf is converted into an osmotically driven trap that closes when an insect triggers sensory trichomes. This study investigates the significance of insect capture for growth of Dionaea at different successional stages after a fire, under conditions where the prey is highly variable in its isotope signature. The contribution of insect-derived nitrogen (N) was estimated using the natural abundance of 15N. In contrast to previous 15N studies on carnivorous plants, the problem emerges that delta15N values of prey insects ranged between -4.47 per thousand (grasshoppers) and +7.21 per thousand (ants), a range that exceeds the delta15N values of non carnivorous reference plants (-4.2 per thousand) and soils (+3 per thousand). Thus, the isotope-mixing model used by Shearer and Kohl to estimate the amount of insect-derived N is not applicable. In a novel approach, the relationships of delta15N values of different organs with delta15N of trapping leaves were used to estimate N partitioning within the plant. It is estimated that soon after fire approximately 75% of the nitrogen is obtained from insects, regardless of plant size or developmental stage. The estimates are verified by calculating the average isotope signatures of insects from an isotope mass balance and comparing this with the average measured delta15N values of insects. It appears that for Dionaea to survive and reach the flowering stage, seedlings must first reach the 6th-leaf rosette stage, in which trap surface area nearly doubles and facilitates the capture of large insects. Large amounts of nitrogen thus made available to plants may facilitate an enhanced growth rate and the progressive production of additional large traps. Dionaea reaches a maximum abundance after fire when growth of the competing vegetation is suppressed. About 10 years after fire, when grasses and shrubs recover, Dionaea becomes overtopped by other species. This would not only reduce carbon assimilation but also the probability of catching larger prey. The amount of insect-derived nitrogen decreases to 46%, and Dionaea becomes increasingly dependent on N-supply from the soil. Competition for both light and N may cause the near disappearance of Dionaea in older stages of the fire succession.

Animals↗

Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps.

The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.

Acid Phosphatase↗

Venus flytraps' metabolome analysis discloses the metabolic fate of prey animal foodstock.

Carnivorous plants such as the Venus flytrap Dionaea muscipula survive in nutrient-poor habitats by attracting and consuming animals. Upon deflection of the touch-sensitive trigger hairs, the trap closes instantly. Panicking prey repeatedly collides with trigger hairs, which activate the endocrine system: mechano- and chemosensors translate the information on the prey's nature, size, and activity into jasmonate-dependent lytic enzyme secretion. This digestive fluid gradually degrades its exoskeleton and internal tissues. The released substances are absorbed by glands covering the inner trap surface. To understand Dionaea's modification of metabolism upon prey consumption, we compared the metabolic profiles associated with secretion and insect feeding. In favor of digestive enzyme secretion, the abundance of most amino acids decreased after JA-stimulation without prey present. By contrast, insect feeding resulted in an increase in almost all amino acids within the trap. In agreement with the export of prey-derived nitrogen, the abundance of certain amino acids also increased in the petiole. In response to feeding with urea, chitin, nucleic acids, or phospholipids, the amino acid profile remained relatively unchanged. This might indicate that the alterations in the Venus flytrap's metabolism depend both on the type of substance and on its amount.

Droseraceae↗

Some features of secretory systems in plants.

Recent work on secretion in plants is reviewed, with emphasis on the anatomy and physiology of root cap cells in higher plants, the stalked glands of Drosera capensis, and the secretory mechanism of Dionaea muscipula. Cells of the root cap of higher plants switch from a geo-perceptive role to one of mucilage secretion at maturation. Features of this process, the role of the Golgi and the pathway for mucilage distribution are reviewed. In contrast, the stalked glands of the leaves of Drosera capensis are much longer lived and have a complex anatomy. The mechanisms for mucilage secretion, protein absorption and the role of the cell membranes in the internal secretion of the protein are described, using data from X-ray microscopv. The secretion of fluid and protein by Dionaea is stimulated by various nitrogen-containing compounds. Uric acid, often excreted by captured insects, is particularly effective in this respect.

Ammonia↗

How the Venus flytrap snaps.

The rapid closure of the Venus flytrap (Dionaea muscipula) leaf in about 100 ms is one of the fastest movements in the plant kingdom. This led Darwin to describe the plant as "one of the most wonderful in the world". The trap closure is initiated by the mechanical stimulation of trigger hairs. Previous studies have focused on the biochemical response of the trigger hairs to stimuli and quantified the propagation of action potentials in the leaves. Here we complement these studies by considering the post-stimulation mechanical aspects of Venus flytrap closure. Using high-speed video imaging, non-invasive microscopy techniques and a simple theoretical model, we show that the fast closure of the trap results from a snap-buckling instability, the onset of which is controlled actively by the plant. Our study identifies an ingenious solution to scaling up movements in non-muscular engines and provides a general framework for understanding nastic motion in plants.

Biomechanical Phenomena↗

Receptor response in Venus's fly-trap.

The insect-trapping movement of the plant Dionaea muscipula (Venus's fly-trap) is mediated by the stimulation of mechanosensory hairs located on the surface of the trap. It is known that stimulation of the hairs is followed by action potentials which are propagated over the surface of the trap. It has been reported that action potentials always precede trap closure. The occurrence of non-propagated receptor potentials is reported here. Receptor potentials always precede the action potentials. The receptor potential appears to couple the mechanical stimulation step to the action potential step of the preying sequence. Receptor potentials elicited by mechanical stimulation of a sensory hair were measured by using the hair as an integral part of the current-measuring path. The tip of the hair was cut off exposing the medullary tissue; this provided a natural extension of the measuring electrode into the receptor region at the base of the hair. A measuring pipette electrode was slipped over the cut tip of the hair. Positive and negative receptor potentials were measured. Evidence is presented which supports the hypothesis that the positive and negative receptor potentials originate from independent sources. An analysis is made of (a) the relation of the parameters of mechanical stimuli to the magnitude of the receptor potential, and (b) the relation of the receptor potentials to the action potential. The hypothesis that the positive receptor potential is the generator of the action potential is consistent with these data.

Electrophysiology↗

The Role of ATP in Mechanically Stimulated Rapid Closure of the Venus's Flytrap.

When the midribs of untreated traps of Dionaea muscipula are frozen in liquid nitrogen after rapid closure, they contain significantly less ATP than those frozen before closure. Exogenous ATP causes a significant increase in the rate of mechanically stimulated trap closure. Illuminated traps close faster than those kept in the dark. The traps of plants placed in 100% O(2) close much faster than do air controls, while 100% CO(2) inhibits closure. It is concluded that ATP is probably the native source of potential energy for contraction of the trap's midrib, and that if the endogenous ATP titer is increased by oxidative phosphorylation or an exogenous source, the trap will close faster.

Journal Article↗