Photosynthetic cyclic electron transport provides ATP for homeostasis during trap closure in Dionaea muscipula.

BACKGROUND AND AIMS: The processes connected with prey capture and the early consumption of prey by carnivorous Dionaea muscipula require high amounts of energy. The aim of the present study was to identify processes involved in flytrap energy provision and ATP homeostasis under these conditions. METHODS: We determined photosynthetic CO2 uptake and chlorophyll fluorescence as well as the dynamics of ATP contents in the snap traps upon closure with and without prey. KEY RESULTS: The results indicate that upon prey capture, a transient switch from linear to cyclic electron transport mediates a support of ATP homeostasis. Beyond 4 h after prey capture, prey resources contribute to the traps' ATP pool and, 24 h after prey capture, export of prey-derived resources to other plant organs may become preferential and causes a decline in ATP contents. CONCLUSIONS: Apparently, the energy demand of the flytrap for prey digestion and nutrient mining builds on both internal and prey-derived resources.

Jasmonate signalling in carnivorous plants: copycat of plant defence mechanisms.

The lipid-derived jasmonate phytohormones (JAs) regulate a wide spectrum of physiological processes in plants such as growth, development, tolerance to abiotic stresses, and defence against pathogen infection and insect attack. Recently, a new role for JAs has been revealed in carnivorous plants. In these specialized plants, JAs can induce the formation of digestive cavities and regulate enzyme production in response to different stimuli from caught prey. Appearing to be a new function for JAs in plants, a closer look reveals that the signalling pathways involved resemble known signalling pathways from plant defence mechanisms. Moreover, the digestion-related secretome of carnivorous plants is composed of many pathogenesis-related (PR) proteins and low molecular weight compounds, indicating that the plant carnivory syndrome is related to and has evolved from plant defence mechanisms. This review describes the similarities between defence and carnivory. It further describes how, after recognition of caught insects, JAs enable the carnivorous plants to digest and benefit from the prey. In addition, a causal connection between electrical and jasmonate signalling is discussed.

Carnivorous leaves from Baltic amber.

The fossil record of carnivorous plants is very scarce and macrofossil evidence has been restricted to seeds of the extant aquatic genus Aldrovanda of the Droseraceae family. No case of carnivorous plant traps has so far been reported from the fossil record. Here, we present two angiosperm leaves enclosed in a piece of Eocene Baltic amber that share relevant morphological features with extant Roridulaceae, a carnivorous plant family that is today endemic to the Cape flora of South Africa. Modern Roridula species are unique among carnivorous plants as they digest prey in a complex mutualistic association in which the prey-derived nutrient uptake depends on heteropteran insects. As in extant Roridula, the fossil leaves possess two types of plant trichomes, including unicellular hairs and five size classes of multicellular stalked glands (or tentacles) with an apical pore. The apices of the narrow and perfectly tapered fossil leaves end in a single tentacle, as in both modern Roridula species. The glandular hairs of the fossils are restricted to the leaf margins and to the abaxial lamina, as in extant Roridula gorgonias. Our discovery supports current molecular age estimates for Roridulaceae and suggests a wide Eocene distribution of roridulid plants.

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid.

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.

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.

Widespread paleopolyploidy, gene tree conflict, and recalcitrant relationships among the carnivorous Caryophyllales.

PREMISE OF STUDY: The carnivorous members of the large, hyperdiverse Caryophyllales (e.g., Venus flytrap, sundews, and Nepenthes pitcher plants) represent perhaps the oldest and most diverse lineage of carnivorous plants. However, despite numerous studies seeking to elucidate their evolutionary relationships, the early-diverging relationships remain unresolved. METHODS: To explore the utility of phylogenomic data sets for resolving relationships among the carnivorous Caryophyllales, we sequenced 10 transcriptomes, including all the carnivorous genera except those in the rare West African liana family Dioncophyllaceae. We used a variety of methods to infer the species tree, examine gene tree conflict, and infer paleopolyploidy events. KEY RESULTS: Phylogenomic analyses supported the monophyly of the carnivorous Caryophyllales, with a crown age of 68-83 million years. In contrast to previous analyses, we recovered the remaining noncore Caryophyllales as nonmonophyletic, although the node supporting this relationship contained a significant amount of gene tree discordance. We present evidence that the clade contains at least seven independent paleopolyploidy events, previously unresolved nodes from the literature have high levels of gene tree conflict, and taxon sampling influences topology even in a phylogenomic data set, regardless of the use of coalescent or supermatrix methods. CONCLUSIONS: Our data demonstrate the importance of carefully considering gene tree conflict and taxon sampling in phylogenomic analyses. Moreover, they provide a remarkable example of the propensity for paleopolyploidy in angiosperms, with at least seven such events in a clade of less than 2500 species.

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.

Proof of anthocyanins in the carnivorous plant genus Nepenthes.

Yellow to red colored betalains are a chemotaxonomic feature of Caryophyllales, while in most other plant taxa, anthocyanins are responsible for these colors. The carnivorous plant family Nepenthaceae belongs to Caryophyllales; here, red-pigmented tissues seem to attract insect prey. Strikingly, the chemical nature of red color in Nepenthes has never been elucidated. Although belonging to Caryophyllales, in Nepenthes, some molecular evidence supports the presence of anthocyanins rather than betalains. However, there was previously no direct chemical proof of this. Using ultra-high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry, we identified cyanidin glycosides in Nepenthes species and tissues. Further, we reveal the existence of a complete set of constitutively expressed anthocyanin biosynthetic genes in Nepenthes. Thus, here we finally conclude the long-term open question regarding red pigmentation in Nepenthaceae.

Nepenthes × ventrata Transcriptome Profiling Reveals a Similarity Between the Evolutionary Origins of Carnivorous Traps and Floral Organs.

The emergence of the carnivory syndrome and traps in plants is one of the most intriguing questions in evolutionary biology. In the present study, we addressed it by comparative transcriptomics analysis of leaves and leaf-derived pitcher traps from a predatory plant Nepenthes ventricosa × Nepenthes alata. Pitchers were collected at three stages of development and a total of 12 transcriptomes were sequenced and assembled de novo. In comparison with leaves, pitchers at all developmental stages were found to be highly enriched with upregulated genes involved in stress response, specification of shoot apical meristem, biosynthesis of sucrose, wax/cutin, anthocyanins, and alkaloids, genes encoding digestive enzymes (proteases and oligosaccharide hydrolases), and flowering-related MADS-box genes. At the same time, photosynthesis-related genes in pitchers were transcriptionally downregulated. As the MADS-box genes are thought to be associated with the origin of flower organs from leaves, we suggest that Nepenthes species could have employed a similar pathway involving highly conserved MADS-domain transcription factors to develop a novel structure, pitcher-like trap, for capture and digestion of animal prey during the evolutionary transition to carnivory. The data obtained should clarify the molecular mechanisms of trap initiation and development and may contribute to solving the problem of its emergence in plants.

Dynamics of amino acid redistribution in the carnivorous Venus flytrap (Dionaea muscipula) after digestion of 13 C/15 N-labelled prey.

Amino acids represent an important component in the diet of the Venus flytrap (Dionaea muscipula), and supply plants with much needed nitrogen resources upon capture of insect prey. Little is known about the significance of prey-derived carbon backbones of amino acids for the success of Dionaea's carnivorous life-style. The present study aimed at characterizing the metabolic fate of 15 N and 13 C in amino acids acquired from double-labeled insect powder. We tracked changes in plant amino acid pools and their δ13 C- and δ15 N-signatures over a period of five weeks after feeding, as affected by contrasting feeding intensity and tissue type (i.e., fed and non-fed traps and attached petioles of Dionaea). Isotope signatures (i.e., δ13 C and δ15 N) of plant amino acid pools were strongly correlated, explaining 60% of observed variation. Residual variation was related to contrasting effects of tissue type, feeding intensity and elapsed time since feeding. Synthesis of nitrogen-rich transport compounds (i.e., amides) during peak time of prey digestion increased 15 N- relative to 13 C- abundances in amino acid pools. After completion of prey digestion, 13 C in amino acid pools was progressively exchanged for newly fixed 12 C. The latter process was most evident for non-fed traps and attached petioles of plants that had received ample insect powder. We argue that prey-derived amino acids contribute to respiratory energy gain and loss of 13 CO2 during conversion into transport compounds (i.e., 2 days after feeding), and that amino-nitrogen helps boost photosynthetic carbon gain later on (i.e., 5 weeks after feeding).

Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species.

In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea's leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g., proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.