Short-term influence of Cu, Zn, Ni and Cd excess on metabolism, ultrastructure and distribution of elements in lichen Xanthoria parietina (L.) Th. Fr.

Lichens are symbiotic organisms that are very sensitive to heavy metal pollution. However, there is little evidence of how heavy metal pollution affects the physiological status, ultrastructural changes and distribution of elements in the layers of lichen thalli. For this purpose we simulated metal pollution to lichens and studied its impact on Xanthoria parietina. Thalli were treated with the heavy metals Cu, Zn, Ni, Cd in the form of sulfates at concentrations of 100µM and 500µM during 24, 48 and 72h. Untreated lichens served as controls. We assessed the status of physiological parameters (fluorescence and integrity of chlorophyll a, content of soluble proteins and ergosterol), ultrastructural changes, especially to the photobiont, and the distribution of elements in the layers of thalli in relation to treatment with heavy metals. We found positive correlations between the content of all tested heavy metals and the physiological response. We assessed the toxicity of the selected metals as follows: Cd >= Cu >= Ni > Zn, based on the effects on the photobiont layer in the lichen thallus and physiological measurements.

Plant growth promotion by inoculation with selected bacterial strains versus mineral soil supplements.

In the process of remediation of mine sites, the establishment of a vegetation cover is one of the most important tasks. This study tests two different approaches to manipulate soil properties in order to facilitate plant growth. Mine waste from Ingurtosu, Sardinia, Italy rich in silt, clay, and heavy metals like Cd, Cu, and Zn was used in a series of greenhouse experiments. Bacteria with putative beneficial properties for plant growth were isolated from this substrate, propagated and consortia of ten strains were used to inoculate the substrate. Alternatively, sand and volcanic clay were added. On these treated and untreated soils, seeds of Helianthus annuus, of the native Euphorbia pithyusa, and of the grasses Agrostis capillaris, Deschampsia flexuosa and Festuca rubra were germinated, and the growth of the seedlings was monitored. The added bacteria established well under all experimental conditions and reduced the extractability of most metals. In association with H. annuus, E. pithyusa and D. flexuosa bacteria improved microbial activity and functional diversity of the original soil. Their effect on plant growth, however, was ambiguous and usually negative. The addition of sand and volcanic clay, on the other hand, had a positive effect on all plant species except E. pithyusa. Especially the grasses experienced a significant benefit. The effects of a double treatment with both bacteria and sand and volcanic clay were rather negative. It is concluded that the addition of mechanical support has great potential to boost revegetation of mining sites though it is comparatively expensive. The possibilities offered by the inoculation of bacteria, on the other hand, appear rather limited.

Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.

A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as "proto-carnivores", lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional "carnivorous organ", which can trap a prey, digest it, and finally absorb available nutrients.

Endocytosis and vesicle trafficking during tip growth of root hairs.

The directional elongation of root hairs, "tip growth", depends on the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material. So far, little is known about the dynamics of endocytosis in living root hairs. We analyzed the motile behaviour of vesicles in the apical region of living root hairs of Arabidopsis thaliana and of Triticum aestivum by live cell microscopy. For direct observation of endocytosis and of the fate of endocytic vesicles, we used the fluorescent endocytosis marker dyes FM 1-43 and FM 4-64. Rapid endocytosis was detected mainly in the tip, where it caused a bright fluorescence of the apical cytoplasm. The internalized membranes proceeded through highly dynamic putative early endosomes in the clear zone to larger endosomal compartments in the subapical region that are excluded from the clear zone. The internalized cargo ended up in the dynamic vacuole by fusion of large endosomal compartments with the tonoplast. Before export to these lytic compartments, putative early endosomes remained in the apical zone, where they most probably recycled to the plasma membrane and back into the cytoplasm for more than 30 min. Endoplasmic reticulum was not involved in trafficking pathways of endosomes. Actin cytoskeleton was needed for the endocytosis itself, as well as for further membrane trafficking. The actin-depolymerizing drug latrunculin B modified the dynamic properties of vesicles and endosomes; they became immobilized and aggregated in the tip. Treatment with brefeldin A inhibited membrane trafficking and caused the disappearance of FM-containing vesicles and putative early endosomes from the clear zone; labelled structures accumulated in motile brefeldin A-induced compartments. These large endocytic compartments redispersed upon removal of the drug. Our results hence prove that endocytosis occurs in growing root hairs. We show the localization of endocytosis in the tip and indicate specific endomembrane compartments and their recycling.

Statoliths motions in gravity-perceiving plant cells: does actomyosin counteract gravity?

Statocytes from plant root caps are characterized by a polar arrangement of cell organelles and sedimented statoliths. Cortical microtubules and actin microfilaments contribute to development and maintenance of this polarity, whereas the lack of endoplasmic microtubules and prominent bundles of actin microfilaments probably facilitates sedimentation of statoliths. High-resolution video microscopy shows permanent motion of statoliths even when sedimented. After immunofluorescence microscopy using antibodies against actin and myosin II the most prominent labeling was observed at and around sedimented statoliths. Experiments under microgravity have demonstrated that the positioning of statoliths depends on the external gravitational force and on internal forces, probably exerted by the actomyosin complex, and that transformation of the gravistimulus evidently occurs in close vicinity to the statoliths. These results suggest that graviperception occurs dynamically within the cytoplasm via small-distance sedimentation rather than statically at the lowermost site of sedimentation. It is hypothesized that root cap cells are comparing randomized motions with oriented motions of statoliths and thereby perceiving gravity.

Interactive computer-assisted position acquisition procedure designed for the analysis of organelle movement in pollen tubes.

An interactive computer-assisted video microscopy method has been developed for the acquisition of extensive data on the sequential positions of pollen tube organelles, which cannot be automatically tracked using geometric or motion patterns. The method consists of video microscopy, analog and digital contrast enhancement, digital time-lapsing of the images, and interactive selection of positions in a coordinate system corresponding to the cell shape and real size. Data on 15,000 positions acquired with this method have been used to make quantitative analyses of the movement patterns of the organelles. From these analyses and the reconstruction of 900 trajectories, it appears that movements are random in the tip of the pollen tube but become more directed in distal regions of the cell, indicating an increase in axial arrangement of the actin filaments. (All custom-made software is available from the authors on request.)

Actin-based vesicle dynamics and exocytosis during wound wall formation in characean internodal cells.

Characean internodal cells readily form wound walls upon local membrane damage. In the present study we documented the dynamics of vesicles involved in wound wall secretion and compared them with actin organization in equivalent cells using immunofluorescence. Single exocytotic events (spreading of vesicle contents) could be visualized using image enhancement by video microscopy. In control unwounded cells vesicles moved unidirectionally along parallel actin bundles and rarely contacted the plasma membrane. The wound response started with (1) local inhibition of active cytoplasmic streaming (unidirectional movements) due to inactivation, depolymerization, or mechanical displacement of the subcortical actin bundles. Accordingly, vesicles performed only oscillating motions and moved slowly with the same velocity and direction as passive endoplasmic flow. (2) Several minutes after wounding, vesicles started to perform random saltatory movements with frequently changing velocities, punctuated by oscillating motion and periods of immobility (docking) at the plasma membrane. Vesicle trajectories correlated with a fine-meshed actin network at the wound site. (3) Several hours after wounding, vesicles moved again unidirectionally along regenerated subcortical actin bundles. Spreading of vesicles (vesicle contents) was observed during wound wall formation, i.e., during the period of saltatory movements when vesicles had access to the plasma membrane. Dependent on the type of wound wall being secreted, three variants could be distinguished: (1) slow and continuous spreading over a time period of several seconds up to 30 min near the plasma membrane, (2) fast spreading within 80 ms inside an already formed wound wall, and/or (3) fast spreading at the plasma membrane. We conclude from our study that wounding-induced changes in vesicle dynamics are due to transient reorganization of the actin cytoskeleton from parallel bundles to a fine-meshed network. Furthermore, our results indicate that spreading of vesicle contents varies considerably with time and may be delayed by vesicle docking and/or discharge.