Alternative or cytochrome? Respiratory pathways in traps of aquatic carnivorous bladderwort Utricularia reflexa.

Carnivorous plants of the genus Utricularia (bladderwort) form modified leaves into suction bladder traps. The bladders are metabolically active plant tissue with high rates of mitochondrial respiration (RD). In general, plants possess two mitochondrial electron transport pathways to reduce oxygen to water: cytochrome and an alternative. Due to the high metabolic rate in the bladders, it is tempting to assume that the bladders prefer the cytochrome c oxidative pathway. Surprisingly, we revealed that alternative oxidase (AOX), which yields only a little ATP, is much more abundant in the bladders of Utricularia reflexa in comparison with the shoots. This pattern is similar to the carnivorous plants with passive pitcher traps (e.g. Sarracenia, Nepenthes) and seems to be widespread across many carnivorous taxa. The exact role of AOX in the traps of carnivorous plants remains to be investigated.

Diethyl ether anesthesia induces transient cytosolic [Ca2+] increase, heat shock proteins, and heat stress tolerance of photosystem II in Arabidopsis.

General volatile anesthetic diethyl ether blocks sensation and responsive behavior not only in animals but also in plants. Here, using a combination of RNA-seq and proteomic LC-MS/MS analyses, we investigated the effect of anesthetic diethyl ether on gene expression and downstream consequences in plant Arabidopsis thaliana. Differential expression analyses revealed reprogramming of gene expression under anesthesia: 6,168 genes were upregulated, 6,310 genes were downregulated, while 9,914 genes were not affected in comparison with control plants. On the protein level, out of 5,150 proteins identified, 393 were significantly upregulated and 227 were significantly downregulated. Among the highest significantly downregulated processes in etherized plants were chlorophyll/tetrapyrrole biosynthesis and photosynthesis. However, measurements of chlorophyll a fluorescence did not show inhibition of electron transport through photosystem II. The most significantly upregulated process was the response to heat stress (mainly heat shock proteins, HSPs). Using transgenic A. thaliana expressing APOAEQUORIN, we showed transient increase of cytoplasmic calcium level [Ca2+]cyt in response to diethyl ether application. In addition, cell membrane permeability for ions also increased under anesthesia. The plants pre-treated with diethyl ether, and thus with induced HSPs, had increased tolerance of photosystem II to subsequent heat stress through the process known as cross-tolerance or priming. All these data indicate that diethyl ether anesthesia may partially mimic heat stress in plants through the effect on plasma membrane.

Anaesthetic diethyl ether impairs long-distance electrical and jasmonate signaling in Arabidopsis thaliana.

General volatile anaesthetics (GVA) inhibit electrical signal propagation in animal neurons. Although plants do not have neurons, they generate and propagate electrical signals systemically from a local damaged leaf to neighbouring leaves. This systemic electrical signal propagation is mediated by ligand-gated glutamate receptor-like (GLR) channels. Here, we investigated the effect of GVA diethyl ether on the systemic electrical and further downstream responses in Arabidopsis thaliana. We monitored electrical signals, cytoplasmic Ca2+ level ([Ca2+]cyt), ultra-weak photon emission, amino acid contents, phytohormone response as well as gene expression in response to heat wounding during diethyl ether anaesthesia. We found complete suppression of electrical and [Ca2+]cyt signal propagation from damaged leaf to neighbouring systemic leaves upon diethyl ether treatment. Concomitantly, jasmonates (JAs) did not accumulate and expression of JA-responsive genes (AOS, OPR3, JAZ10) was not detected in systemic leaves. However local damaged leaves still showed increased [Ca2+]cyt and accumulated high level of JAs and JA-inducible transcripts. An exogenously added GLR ligand, L-glutamate, was not able to trigger Ca2+ wave in etherized plants indicating that GLRs are targeted by diethyl ether, but not specifically. The fact that GVA inhibit electrical signal propagation not only in animals but also in plants is intriguing. However, the cellular response is completely blocked only in systemic leaves; the local damaged leaf still senses damaging stimuli.

Contrasting effect of prey capture on jasmonate accumulation in two genera of aquatic carnivorous plants (Aldrovanda, Utricularia).

Terrestrial carnivorous plants of genera Drosera, Dionaea and Nepenthes within the order Caryophyllales employ jasmonates for the induction of digestive processes in their traps. Here, we focused on two aquatic carnivorous plant genera with different trapping mechanism from distinct families and orders: Aldrovanda (Droseraceae, Caryophyllales) with snap-traps and Utricularia (Lentibulariaceae, Lamiales) with suction traps. Using phytohormone analyses and simple biotest, we asked whether the jasmonates are involved in the activation of carnivorous response similar to that known in traps of terrestrial genera of Droseraceae (Drosera, Dionaea). The results showed that Utricularia, in contrast with Aldrovanda, does not use jasmonates for activation of carnivorous response and is the second genus in Lamiales, which has not co-opted jasmonate signalling for botanical carnivory. On the other hand, the nLC-MS/MS analyses revealed that both genera secreted digestive fluid containing cysteine protease homologous to dionain although the mode of its regulation may differ. Whereas in Utricularia the cysteine protease is present constitutively in digestive fluid, it is induced by prey and exogenous application of jasmonic acid in Aldrovanda.

Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula × Tina.

Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula × Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. × Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.

Anaesthesia with diethyl ether impairs jasmonate signalling in the carnivorous plant Venus flytrap (Dionaea muscipula).

BACKGROUND AND AIMS: General anaesthetics are compounds that induce loss of responsiveness to environmental stimuli in animals and humans. The primary site of action of general anaesthetics is the nervous system, where anaesthetics inhibit neuronal transmission. Although plants do not have neurons, they generate electrical signals in response to biotic and abiotic stresses. Here, we investigated the effect of the general volatile anaesthetic diethyl ether on the ability to sense potential prey or herbivore attacks in the carnivorous plant Venus flytrap (Dionaea muscipula). METHODS: We monitored trap movement, electrical signalling, phytohormone accumulation and gene expression in response to the mechanical stimulation of trigger hairs and wounding under diethyl ether treatment. KEY RESULTS: Diethyl ether completely inhibited the generation of action potentials and trap closing reactions, which were easily and rapidly restored when the anaesthetic was removed. Diethyl ether also inhibited the later response: jasmonic acid (JA) accumulation and expression of JA-responsive genes (cysteine protease dionain and type I chitinase). However, external application of JA bypassed the inhibited action potentials and restored gene expression under diethyl ether anaesthesia, indicating that downstream reactions from JA are not inhibited. CONCLUSIONS: The Venus flytrap cannot sense prey or a herbivore attack under diethyl ether treatment caused by inhibited action potentials, and the JA signalling pathway as a consequence.

Taste for protein: Chemical signal from prey stimulates enzyme secretion through jasmonate signalling in the carnivorous plant Venus flytrap.

Hunting cycle of the carnivorous plant Venus flytrap (Dionaea muscipula Ellis) is comprised of mechanism for rapid trap closure followed by slow hermetical sealing and activation of gene expression responsible for digestion of prey and nutrient uptake. In the present study, we focus on the late phase of Venus's flytrap hunting cycle when mechanical stimulation of the prey ceases and is replaced by chemical cues. We used two nitrogen-rich compounds (chitin and protein) in addition to mechanostimulation to investigate the electrical and jasmonate signalling responsible for induction of enzyme activities. Chemical stimulation by BSA protein and chitin did not induce any additional spontaneous action potentials (APs). However, chemical stimulation by protein induced the highest levels of jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) as well as the expression of studied gene encoding a cysteine protease (dionain). Although chitin is probably the first chemical agent which is in direct contact with digestive glands, presence of protein in the secured trap mimics the presence of insect prey best.

Triggering a false alarm: wounding mimics prey capture in the carnivorous Venus flytrap (Dionaea muscipula).

In the carnivorous plant Venus flytrap (Dionaea muscipula), the sequence of events after prey capture resembles the well-known plant defence signalling pathway in response to pathogen or herbivore attack. Here, we used wounding to mimic prey capture to show the similarities and differences between botanical carnivory and plant defence mechanisms. We monitored movement, electrical signalling, jasmonate accumulation and digestive enzyme secretion in local and distal (systemic) traps in response to prey capture, the mechanical stimulation of trigger hairs and wounding. The Venus flytrap cannot discriminate between wounding and mechanical trigger hair stimulation. Both induced the same action potentials, rapid trap closure, hermetic trap sealing, the accumulation of jasmonic acid (JA) and its isoleucine conjugate (JA-Ile), and the secretion of proteases (aspartic and cysteine proteases), phosphatases and type I chitinase. The jasmonate accumulation and enzyme secretion were confined to the local traps, to which the stimulus was applied, which correlates with the propagation of electrical signals and the absence of a systemic response in the Venus flytrap. In contrast to plant defence mechanisms, the absence of a systemic response in carnivorous plant may represent a resource-saving strategy. During prey capture, it could be quite expensive to produce digestive enzymes in the traps on the plant without prey.