On the mechanism of trap closure of Venus flytrap (Dionaea muscipula Ellis)

The rapid trap closure of Dionaea muscinula Ellis has been explained by either a loss of turgor pressure of the upper epidermis, which should thus become flexible, or by a sudden acid-induced wall loosening of the motor cells. According to our experiments both explanations are doubtful. Objections against the turgor mechanism come from the determination by extracellular measurements from the upper epidermis of action-potential amplitudes before and after trap closure. Neither time course nor amplitude of the action potentials are altered by trap closure. In contrast a rise in the apoplastic concentration of K⁺ or Na⁺, which are the only ions present in the trap in osmotically significant concentrations, from 1 to 10 mM reduces the action-potential amplitudes by 25% and 15%, respectively. Furthermore, after trap closure the upper epidermal cells retain a considerable cell sap osmolality of 0.41 mol·kg^-1 which equals that of the mesophyll cells as determined by incipient plasmolysis. A sudden cell-wall acidification causing movement is improbable since an acidification of the apoplast from pH 6 to pH 4 reduces action-potential amplitudes by 33% whereas the amplitudes measured extracellylarly from the mesophyll and lower epidermis remain unchanged by trap closure. In addition, buffering the apoplast at pH 6 does not prevent movement in traps which have been incised several times from the margin to the midrib to facilitate buffer diffusion into the mesophyll. Even an alkalinization of cell walls of plasmolysed leaf segments to pH 9 does not prevent considerable extensions of the mesophyll and subsequent movement of the specimens during deplasmolysis.These experiments make it very likely that the mesophyll cells are already extensible but are kept compressed in the open trap, thus developing tissue tension. The mechanism which prevents their extension as long as the trap is open can so far only be explained for traps which have been paralysed by a long-term incubation in 1 mM La³⁺. Leaf strips taken from stimulated, closed traps, comprising the lower epidermis and some mesophyll, prove to be highly extensible if they are stretched perpendicular to the midrib on a constant-load extensiometer. By contrast, strips taken from the lower side of paralysed traps are as rigid as those from the upper side of both stimulated and paralysed traps. From observations of semithin cross sections in a polarizing microscope, it is concluded that the extensibilities of these tissue strips are mainly determined by the cell walls of the upper epidermis plus a layer of adjacent mesophyll and by the lower epidermis, respectively, since these are the only cell walls with a preferential microfibril orientation in the direction of the applied stress.Abbreviations E_m membrane potential - E_s surface potential - Mes 2-(N-morpholino)ethanesulfonic acid - Tris 2-amino-2(hydroxymethyl)-1,3-propanediol     

On the mechanism underlying photosynthetic limitation upon trigger hair irritation in the carnivorous plant Venus flytrap (Dionaea muscipula Ellis)

Mechanical stimulation of trigger hairs on the adaxial surface of the trap of Dionaea muscipula leads to the generation of action potentials and to rapid leaf movement. After rapid closure secures the prey, the struggle against the trigger hairs results in generation of further action potentials which inhibit photosynthesis. A detailed analysis of chlorophyll a fluorescence kinetics and gas exchange measurements in response to generation of action potentials in irritated D. muscipula traps was used to determine the 'site effect' of the electrical signal-induced inhibition of photosynthesis. Irritation of trigger hairs and subsequent generation of action potentials resulted in a decrease in the effective photochemical quantum yield of photosystem II (Φ(PSII)) and the rate of net photosynthesis (A(N)). During the first seconds of irritation, increased excitation pressure in photosystem II (PSII) was the major contributor to the decreased Φ(PSII). Within ～1?min, non-photochemical quenching (NPQ) released the excitation pressure at PSII. Measurements of the fast chlorophyll a fluorescence transient (O-J-I-P) revealed a direct impact of action potentials on the charge separation-recombination reactions in PSII, although the effect seems to be small rather than substantial. All the data presented here indicate that the main primary target of the electrical signal-induced inhibition of photosynthesis is the dark reaction, whereas the inhibition of electron transport is only a consequence of reduced carboxylation efficiency. In addition, the study also provides valuable data confirming the hypothesis that chlorophyll a fluorescence is under electrochemical control.     

Trap closure and prey retention in Venus flytrap (Dionaea muscipula) temporarily reduces photosynthesis and stimulates respiration

Background and Aims

The carnivorous plant Venus flytrap (Dionaea muscipula) produces a rosette of leaves: each leaf is divided into a lower part called the lamina and an upper part, the trap, with sensory trigger hairs on the adaxial surface. The trap catches prey by very rapid closure, within a fraction of a second of the trigger hairs being touched twice. Generation of action potentials plays an important role in closure. Because electrical signals are involved in reduction of the photosynthetic rate in different plant species, we hypothesized that trap closure and subsequent movement of prey in the trap will result in transient downregulation of photosynthesis, thus representing the energetic costs of carnivory associated with an active trapping mechanism, which has not been previously described.

Methods

Traps were enclosed in a gas exchange cuvette and the trigger hairs irritated with thin wire, thus simulating insect capture and retention. Respiration rate was measured in darkness (R_D). In the light, net photosynthetic rate (A_N), stomatal conductance (g_s) and intercellular CO₂ concentration (c_i) were measured, combined with chlorophyll fluorescence imaging. Responses were monitored in the lamina and trap separately.

Key Results

Irritation of trigger hairs resulted in decreased A_N and increased R_D, not only immediately after trap closure but also during the subsequent period when prey retention was simulated in the closed trap. Stomatal conductance remained stable, indicating no stomatal limitation of A_N, so c_i increased. At the same time, the effective quantum yield of photosystem II (Φ_PSII) decreased transiently. The response was confined mainly to the digestive zone of the trap and was not observed in the lamina. Stopping mechanical irritation resulted in recovery of A_N, R_D and Φ_PSII.

Conclusions

We put forward the first experimental evidence for energetic demands and carbon costs during insect trapping and retention in carnivorous plants, providing a new insight into the cost/benefit model of carnivory.     

The dynamics of H+ efflux from the trap lobes of Dionaea muscipula Ellis (Venus's flytrap)

Abstract. H⁺ efflux from the trap lobes of Dionaea muscipula Ellis (Venus's flytrap) was measured in vitro. FC, IAA, and 2,4-D markedly increase the rate of H⁺ efflux within minutes of their addition to the incubation medium whereas ABA and DES cause the rate to decrease. Consequently, the H⁺ efflux mechanism of Dionaea is considered to be similar to the H⁺ extrusion pumps of other higher plants in this respect. However, the H⁺ extrusion mechanism of Dionaea may be unusual in that long-term exposure of the trap lobes to known secretion elicitors— bactopeptone, NH₄⁺, Na ⁺, urea, thiourea, glycine or xanthine—also causes a large increase in the steady-state rate of H⁺ efflux from the trap lobes. Since the observed H⁺ effluxes primarily correspond to the adaxial surface of the trap lobes and show similar time- and secretion elicitor-dependencies to the responses seen in situ, it appears that the H⁺ effluxes measured in vitro bear a direct relationship to those observed in the intact, actively secreting plant. Three of the secretion elicitors that were tested— K⁺, NH₄⁺, and urea—have rapid effects on the rate of H⁺ extrusion in addition to their long-term effects. K⁺ and NH₄⁺ cause a rapid acceleration of H⁺ efflux whereas urea causes a rapid deceleration or, at high external concentrations, reversal of the net flux. The effect of K⁺ is inferred to result from K⁺ -H⁺ exchange between the tissue and bathing medium. Studies with structural analogues of NH₄⁺ and urea and inhibitors of the assimilation of reduced nitrogen suggest that the effects of NH₄⁺ and urea result from the pH-perturbing consequences of their metabolism subsequent to their absorption. These effects are considered to be auxiliary to the elicitation of secretion. It is proposed that H⁺ efflux from the trap lobes is mediated by a K⁺-H⁺ exchange mechanism, the activity of which is modified by long-term exposure to secretion elicitors and/or short-term exposure to factors which alter the availability of endogenous H⁺ ions.     

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.     

Abundance of Cysteine Endopeptidase Dionain in Digestive Fluid of Venus Flytrap (Dionaea muscipula Ellis) Is Regulated by Different Stimuli from Prey through Jasmonates

The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey''s movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH₄Cl, KH₂PO₄, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed.     

Source, etiolation and orientation of explants affect in vitro regeneration of Venus fly-trap (Dionaea muscipula)

In vitro culture of Venus fly-trap (Dionaea muscipula) was initiated using flower stalk explants. Activated charcoal was required for bud initiation, but omitted in the subculture of regenerated plantlets. Regenerated plants were subsequently used as explant source for investigations concerning effects of source of tissue, etiolation, orientation and illumination of leaf explants on plant regeneration. Etiolation of source plantlets increased the rate of regeneration from explants and decreased explant failure. Generally, adventitious buds developed at the adaxial side and proximal end of an explant. However, when explants were incubated in the dark, 20–30% of bud initiation occurred at the distal end. The site of shoot regeneration on a leaf explant was affected by both illumination and orientation of explants. Placing an explant adaxial side up resulted in the highest rate of regeneration. The most effective condition for plantlet regeneration was found with etiolated petioles incubated with the adaxial side facing the light. Received: 18 March 1998 / Revision received: 12 August 1998 / Accepted: 7 September 1998     

A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap)

The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme.     

Dionaea D. Solander ex J. Ellis (Droseraceae): notes on the nomenclature and typification of Venus's Fly-trap

NELSON, E. C, 1989. Dionaea Solander ex J. Ellis (Droseraceae): Notes on the nomenclature and typification of Venus's Fly-trap. The nomenclatural history of Venus's flytrap, Dionaea muscipula (Droseraceae) from North America (Carolinas), is discussed. The binomial was first published in a London newspaper at the beginning of September 1768 by John Ellis who credited the generic name to Daniel Solander. A lectotype is designated.     

Chlorophyll a fluorescence induction (Kautsky curve) in a Venus flytrap (Dionaea muscipula) leaf after mechanical trigger hair irritation

This paper describes experiments on transient changes in chlorophyll a fluorescence in traps of the carnivorous plant Venus flytrap (Dionaea muscipula) that occur in association with mechanical stimulation of trigger hairs and propagation of action potentials (APs). The experiments show the following reproducible effects of APs on the fluorescence induction (Kautsky-, or OJIPSMT curve) in a 100 s low intensity light pulse (i) no change in the OJ phase attributed to release of photochemical quenching, (ii) a small enhancement, if at all of increase in the thermal JIP phase, (iii) a two- to threefold deceleration of the fluorescence decline (quenching) during the PSMT phase in the 2–100 s time range, and (iv) a transient 15–50% increase in variable fluorescence within ∼20 s under steady state light condition with, after ∼80 s, a 10% undershoot that reverses in several tens of seconds to the original steady state. The results are discussed in terms of a hypothesis that the fluorescence decline during the SMT phase of the Kautsky induction curve, attributed to NPQ, is caused by the Δμ_H+-driven increase in proton conductance of the CF_o channel of the ATPase during its activation. A signal-transducing role of Ca²⁺ is suggested.     

Characteristics and bending performance of electroactive polymer blend made with cellulose and poly(3-hydroxybutyrate)

This paper describes a novel cellulose/poly(3-hydroxybutyrate) blend based electroactive polymer. The fabrication process, bending actuation test and its characteristics are investigated. To prepare this new EAP, cellulose and PHB were dissolved in trifluoroacetic acid. The solution was cast to form a film followed by depositing thin gold electrode on both sides of the film. The characteristics of the cellulose/PHB film were investigated by Fourier transform infrared spectra, scanning electron microscopy, X-ray diffraction differential scanning calorimetry, tensile test and dynamic mechanical analysis. The bending performance was evaluated in terms of free bending displacement, electrical power consumption output and lifetime test under ambient conditions. Primary results show that this cellulose/PHB blend EAP is less sensitive to humidity and it shows higher bending displacement and longer lifetime than pure cellulose EAP at room humidity condition. These results indicate that this new cellulose/PHB blend EAP has potential for many biomimetic applications.     

Biomimetic zinc oxide replica with structural color using butterfly (Ideopsis similis) wings as templates

Nano-structured colorful zinc oxide (ZnO) replicas were produced using the wings of the Ideopsis similis butterfly as templates. The ZnO replicas we obtained exhibit iridescence, which was clearly observed under an optical microscope (OM). Field emission scanning electron microscope analysis shows that all the microstructure details are maintained faithfully in the ZnO replica. A computer model was established to simulate the diffraction spectral results, which agreed well with the OM images.     

Biomimetic oxidation of Hantzsch 1,4-dihydropyridines with tetra-n-butylammonium periodate catalyzed by tetraphenylporphyrinatomanganese(III) chloride [Mn(TPP)Cl

Efficient oxidation of Hantzsch 1,4-dihydropyridines to their corresponding pyridine derivatives with (Bu(4)N)IO(4) catalyzed by tetraphenylporphyrinatomanganese(III) chloride [Mn(TPP)Cl] is reported. This catalytic system shows high efficiency in the oxidation of 1,4-dihydropyridines at room temperature in the presence of imidazole.     

Biomimetic zinc(II) and cobalt(II) complexes with tri-tert-butoxysilanethiolate and imidazole ligands - Structural and spectroscopic studies

New, heteroleptic zinc and cobalt complexes with tri-tert-butoxysilanethiolate and imidazole co-ligands are characterized by crystal structure studies. The ligands exhibit different coordination modes to Co(II) ions: NOS₂ (with methanol as O-donor ligand) in 2′, NO₂S₂ in 2′′, N₂S₂ in 1, and to Zn(II) ions: N₂S₂ in 3 and N₃S in 4. Complex 2′ is a structural analog of cobalt-substituted active site of alcohol dehydrogenase. All four-coordinate Co(II) and Zn(II) complexes have tetrahedral geometry. Solution and solid state electronic spectra of cobalt(II) complexes are discussed and compared to literature data available for the cobalt-substituted liver alcohol dehydrogenase and sorbitol dehydrogenase. The EPR spectra of all cobalt complexes exhibit at 77 K a characteristic broad signal with g ∼3.6 and 5.6, strongly indicating a high-spin state, S = 3/2, of Co(II) complexes.     

Enzyme reactor with knitted fabric made of poly(vinyl alcohol) superfine filaments

Various supports and bio-reactors have been proposed. Packed bed reactors with polymer material in granular shape are most often employed in both laboratory and industry. But they have a disadvantage related to an increase in pressure drop. We already developed filter paper composed of short cut pieces of superfine filaments (SFF). It shows high performance, but its hydrodynamic resistance increases when substrate solution passes through it. A new type of enzyme reactor equipped with knitted SFF has been proposed. In this reactor, substrate does not pass through the support but flows along the thin channel and parallel to the support. Therefore, it is able to maintain flow rate constant during a considerable period. The productivity of the reactor fairly increases by reducing the thickness of the channel because linear velocity increases with the reduction of the thickness and that contributes to the decrease in mass transfer resistance.     

Biomimetic cooperative interactions of dried cross-linked poly(N-6-aminohexylacrylamide) with binary mixtures of solvent vapors

Biomimetic cooperativity of hydration effect and effect of ethanol favorable for binding of bad organic sorbates were observed for their vapor sorption by cross-linked poly(N-6-aminohexylacrylamide) (PNAHAA) in the absence of liquid phase. The vapor sorption isotherms were determined for these systems by the static method of gas chromatographic headspace analysis at 298 K. The hydration above 0.09-0.13 g of H(2)O/(g of polymer) gives a cooperative increase in the PNAHAA binding affinity for benzene, cyclohexane, dioxane, and propanols up to a level which does not change by further hydration, indicating the polymer antiplasticization. Bad sorbates (dioxane, benzene) were observed to have a biomimetic cooperative influence on the binding of ethanol by the dried PNAHAA. This cooperativity does not occur in ternary systems with good nonhydroxylic sorbate acetonitrile.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

One-dimensional coordination polymers made of transition metal-nitronyl nitroxide radical with dicyanoaurate(I) bridges

Three 1-D transition metal-nitronyl nitroxide radical complexes with dicyanoaurate(I) bridges, [M(NIT3py)₂][Au(CN)₂]₂ [NIT3py = 2-(3′-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, M = Mn, Co, Zn (1-3)], were synthesized and structurally characterized. Three compounds are all isostructural in monoclinic, C2/c space group with Z = 4. The [Au(CN)₂]⁻ anions link [M(NIT3py)₂] units via μ₂-bridging mode, leading to a linear coordination chain. The M(II) ion adopts a distorted octahedral geometry with four N atoms from [Au(CN)₂]⁻ groups and two pyridyl-N atoms from NIT3py ligands. The magnetic behavior shows that the couplings are both weak antiferromagnetic between Mn(II) and NIT3py and between Co(II) and NIT3py.