Quantification of Protein Uptake by Endocytosis in Carnivorous Nepenthales.

Carnivorous plants adsorb prey-derived nutrients partly by endocytosis. This study quantifies endocytosis in Drosophyllum lusitanicum, Drosera capensis, Drosera roseana, Dionaea muscipula and Nepenthes × ventrata. Traps were exposed to 1% fluorescent-labeled albumin (FITC-BSA), and uptake was quantified repeatedly for 64 h. Formation of vesicles started after ≤1 h in adhesive traps, but only after 16 h in species with temporary stomach (D. muscipula and N. × ventrata). In general, there are similarities in the observed species, especially in the beginning stages of endocytosis. Nonetheless, further intracellular processing of endocytotic vesicles seems to be widely different between species. Endocytotic vesicle size increased significantly over time in all species except in D. capensis. Fluorescence intensity of the endocytotic vesicles increased in all species except D. muscipula. After 64 h, estimates for FITC-BSA absorption per gland ranged from 5.9 ± 6.3 ng in D. roseana to 47.8 ± 44.3 ng in N. × ventrata, demonstrating that endocytosis substantially contributes to the adsorption of prey-derived nutrients.

Structural traits of leaf epidermis correspond to metal tolerance in Rumex acetosella populations growing on metal-contaminated soils.

The pseudometallophyte Rumex acetosella L. occupies habitats with normal and high soil concentrations of zinc (Zn), lead (Pb), and copper (Cu). It remains unclear if the plants respond to the toxic metals by altering their morphology and increasing the resilience of their cells. We compared plants growing on soils contaminated with Zn/Pb (populations Terézia, Lintich), or Cu (populations Spania Dolina, Staré Hory), with those from non-contaminated soil (Dúbravka) in Slovakia, and analysed leaf structure, physiology, and metal contents by light and electron microscopy, element localization by energy-dispersive X-ray analysis (EDX) in scanning electron microscope, and by specific fluorescence dyes. In control population, the epidermis of the amphistomatic leaves of R. acetosella contained capitate glandular trichomes, consisting of four head (secretory), two stalk, and two basal cells. The ultrastructure of secretory cells revealed fine wall ingrowths bordered by plasma membrane protruding into the cytoplasm. The metallicolous populations had higher contents of Zn and Cu in the epidermal and glandular cells, and a higher density of both stomata and trichomes. Extensive cell wall labyrinth was present in the trichome secretory cells. Their abnormal number and elevated metal contents might indicate effects of heavy metals, especially of Cu, on mitosis and cell plate formation. Differences in leaf physiology were indicated by significantly higher cytoplasmic tolerance to Zn and Cu in metallicolous populations and by structural properties of glandular heads suggesting secretion of toxic metals. Our findings are suggestive of plant reactions to metal stress, which facilitate the populations to occupy the metal-contaminated sites.

The copper spoil heap Knappenberg, Austria, as a model for metal habitats - Vegetation, substrate and contamination.

Historic mining in the Eastern Alps has left us with a legacy of numerous spoil heaps hosting specific, metal tolerant vegetation. Such habitats are characterized by elevated concentrations of toxic elements but also by high irradiation, a poorly developed substrate or extreme pH of the soil. This study investigates the distribution of vascular plants, mosses and lichens on a copper spoil heap on the ore bearing Knappenberg formed by Prebichl Layers and Werfener Schist in Lower Austria. It serves as a model for discriminating between various ecological traits and their effects on vegetation. Five distinct clusters were distinguished: (1) The bare, metal rich Central Spoil Heap was only colonised by highly resistant specialists. (2) The Northern and (3) Southern Peripheries contained less copper; the contrasting vegetation was best explained by the different microclimate. (4) A forest over acidic bedrock hosted a vegetation overlapping with the periphery of the spoil heap. (5) A forest over calcareous bedrock was similar to the spoil heap with regard to pH and humus content but hosted a vegetation differing strongly to all other habitats. Among the multiple toxic elements at the spoil heap, only Cu seems to exert a crucial influence on the vegetation pattern. Besides metal concentrations, irradiation, humidity, humus, pH and grain size distribution are important for the establishment of a metal tolerant vegetation. The difference between the species poor Northern and the diverse Southern Periphery can be explained by the microclimate rather than by the substrate. All plant species penetrating from the forest into the periphery of the spoil heap originate from the acidic but not from the calcareous bedrock.

Turnover of Phosphatidic Acid through Distinct Signaling Pathways Affects Multiple Aspects of Pollen Tube Growth in Tobacco.

Phosphatidic acid (PA) is an important intermediate in membrane lipid metabolism that acts as a key component of signaling networks, regulating the spatio-temporal dynamics of the endomembrane system and the cytoskeleton. Using tobacco pollen tubes as a model, we addressed the signaling effects of PA by probing the functions of three most relevant enzymes that regulate the production and degradation of PA, namely, phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs). Phylogenetic analysis indicated a highly dynamic evolution of all three lipid-modifying enzymes in land plants, with many clade-specific duplications or losses and massive diversification of the C2-PLD family. In silico transcriptomic survey revealed increased levels of expression of all three PA-regulatory genes in pollen development (particularly the DGKs). Using specific inhibitors we were able to distinguish the contributions of PLDs, DGKs, and LPPs into PA-regulated processes. Thus, suppressing PA production by inhibiting either PLD or DGK activity compromised membrane trafficking except early endocytosis, disrupted tip-localized deposition of cell wall material, especially pectins, and inhibited pollen tube growth. Conversely, suppressing PA degradation by inhibiting LPP activity using any of three different inhibitors significantly stimulated pollen tube growth, and similar effect was achieved by suppressing the expression of tobacco pollen LPP4 using antisense knock-down. Interestingly, inhibiting specifically DGK changed vacuolar dynamics and the morphology of pollen tubes, whereas inhibiting specifically PLD disrupted the actin cytoskeleton. Overall, our results demonstrate the critical importance of all three types of enzymes involved in PA production and degradation, with strikingly different roles of PA produced by the PLD and DGK pathways, in pollen tube growth.

Endocytotic uptake of nutrients in carnivorous plants.

Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.

Structural sterols are involved in both the initiation and tip growth of root hairs in Arabidopsis thaliana.

Structural sterols are abundant in the plasma membrane of root apex cells in Arabidopsis thaliana. They specifically accumulate in trichoblasts during the prebulging and bulge stages and show a polar accumulation in the tip during root hair elongation but are distributed evenly in mature root hairs. Thus, structural sterols may serve as a marker for root hair initiation and growth. In addition, they may predict branching events in mutants with branching root hairs. Structural sterols were detected using the sterol complexing fluorochrome filipin. Application of filipin caused a rapid, concentration-dependent decrease in tip growth. Filipin-complexed sterols accumulated in globular structures that fused to larger FM4-64-positive aggregates in the tip, so-called filipin-induced apical compartments, which were closely associated with the plasma membrane. The plasma membrane appeared malformed and the cytoarchitecture of the tip zone was affected. Trans-Golgi network/early endosomal compartments containing molecular markers, such as small Rab GTPase RabA1d and SNARE Wave line 13 (VTI12), locally accumulated in these filipin-induced apical compartments, while late endosomes, endoplasmic reticulum, mitochondria, plastids, and cytosol were excluded from them. These data suggest that the local distribution and apical accumulation of structural sterols may regulate vesicular trafficking and plasma membrane properties during both initiation and tip growth of root hairs in Arabidopsis.

The abiotic environment of Heliamphora nutans (Sarraceniaceae): pedological and microclimatic observations on Roraima Tepui

The aim of this study was the study of the abiotic environment of the carnivorous pitcher plant Heliamphora nutans (Sarraceniaceae), including the microclimate and the geochemistry of the soil of the growing sites on Roraima Tepui and discuss their relevance within the recent model of carnivorous plant ecology. The soil was peaty and low in nutrients. The microclimate on the site was very balanced, with moderately cool temperatures, a constant high humidity and very low wind speed. Heliamphora was not exposed to any recognizable climatic stress. Previous macroclimatic measurements reflected the growth conditions of Heliamphora only incorrectly, since humidity decreased drastically with height. The apparent conflict with the common model of carnivorous plant ecology was caused by the dense surrounding vegetation. However, the leaf coverage of these non carnivorous plants was too low to cause significant insolation decrease for Heliamphora. Furthermore, the temperature regime of the pitcher fluid was more balanced than the temperature of the leaf. This may improve conditions for the growth of microorganisms in the pitcher fluid that contribute to the degredation of the plant's prey.

Comparing copper resistance in two bryophytes: Mielichhoferia elongata Hornsch. versus Physcomitrella patens Hedw.

The bryophyte Mielichhoferia elongata is known to occur on copper-rich substrate, but the exact resistance level remained to be determined by in vitro experiments. Here, we tested its copper tolerance in graded copper solutions and compared the results to the moss Physcomitrella patens that is not known to inhabit heavy metal sites. Our results confirm the survival of M. elongata in classical resistance experiments of up to 10 mM Cu-ethylenediaminetetraacetic acid (EDTA) solution. Interestingly, P. patens is equally resistant. Cultured on copper-enriched agar plates for over 5 weeks, P. patens survived even higher copper levels of up to 100 mM Cu-EDTA and an increment of growth was detected on all concentrations tested. Obviously, P. patens is able to withstand harmfully high levels of copper in both solution and substrate. In this short communication, we give a detailed description of the growth rates and discuss the results in comparison to other moss species and heavy metals.

An acoustically-driven biochip - impact of flow on the cell-association of targeted drug carriers.

The interaction of targeted drug carriers with epithelial and endothelial barriers in vivo is largely determined by the dynamics of the body fluids. To simulate these conditions in binding assays, a fully biocompatible in vitro model was developed which can accurately mimic a wide range of physiological flow conditions on a thumbnail-format cell-chip. This acoustically-driven microfluidic system was used to study the interaction characteristics of protein-coated particles with cells. Poly(D,L-lactide-co-glycolide) (PLGA) microparticles (2.9 +/- 1 microm) were conjugated with wheat germ agglutinin (WGA-MP, cytoadhesive protein) or bovine serum albumin (BSA-MP, non-specific protein) and their binding to epithelial cell monolayers was investigated under stationary and flow conditions. While mean numbers of 1500 +/- 307 mm(-2) WGA-MP and 94 +/- 64 mm(-2) BSA-MP respectively were detected to be cell-bound in the stationary setup, incubation at increasing flow velocities increasingly antagonized the attachment of both types of surface-modified particles. However, while binding of BSA-MP was totally inhibited by flow, grafting with WGA resulted in a pronounced anchoring effect. This was indicated by a mean number of 747 +/- 241 mm(-2) and 104 +/- 44 mm(-2) attached particles at shear rates of 0.2 s(-1) and 1 s(-1) respectively. Due to the compactness of the fluidic chip which favours parallelization, this setup represents a highly promising approach towards a screening platform for the performance of drug delivery vehicles under physiological flow conditions. In this regard, the flow-chip is expected to provide substantial information for the successful design and development of targeted micro- and nanoparticulate drug carrier systems.

Expanding the menu for carnivorous plants: uptake of potassium, iron and manganese by carnivorous pitcher plants.

Carnivorous plants use animals as fertiliser substitutes which allow them to survive on nutrient deficient soils. Most research concentrated on the uptake of the prey's nitrogen and phosphorus; only little is known on the utilisation of other elements. We studied the uptake of three essential nutrients, potassium, iron and manganese, in three species of carnivorous pitcher plants (Cephalotus follicularis LaBilladiere, Sarracenia purpureaL., Heliamphora nutans Bentham). Using relatively short-lived and gamma-emitting radiotracers, we significantly improved the sensitivity compared to conventional protocols and gained the following results. We demonstrated the uptake of trace elements like iron and manganese. In addition, we found direct evidence for the uptake of potassium into the pitcher tissue. Potassium and manganese were absorbed to virtually 100% if offered in physiological concentrations or below in Cephalotus. Analysis of pitcher fluid collected in the natural habitat showed that uptake was performed here as efficiently as in the laboratory. The absorption of nutrients is an active process depending on living glandular cells in the pitcher epidermis and can be inhibited by azide. Unphysiologically high amounts of nutrients were taken up for a short time, but after a few hours the absorbing cells were damaged, and uptake stopped. Absorption rates of pitcher leaves from plants under controlled conditions varied highly, indicating that each trap is functionally independent. The comparison of minerals in typical prey with the plants' tissues showed that a complete coverage of the plants' needs by prey capture is improbable.