Understanding the mechanisms driving plant defense responses holds the promise to provide new means to reinforce plant defense both through agrochemicals and targeted genetic improvement. The capability to quantify impacts of phytopathogens on subcellular dynamics is particularly important when elucidating the role of specific virulence mechanisms that make contributions toward infection success but do not individually alter disease outcome. Acquiring these data requires an investigator to achieve the successful handling of both plant and microbe prior to observation and an appreciation of the challenges in acquiring images under these conditions. In this chapter we describe a protocol to support the observation of cytoskeletal dynamics surrounding sites of fungal interaction, specifically the powdery mildew Blumeria graminis f.sp. hordei on the surface of Arabidopsis thaliana. Furthermore, we also describe a procedure to expose etiolated (dark-grown) hypocotyls to a molecular pattern to activate defense responses in the absence of a phytopathogen with the aim of observing localized actin-dependent trafficking.

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Cell wall appositions (CWAs) are produced reactively by the plant immune system to arrest microbial invasion through the local inversion of plant cell growth. This process requires the controlled invagination of the plasma membrane (PM) in coordination with the export of barrier material to the volume between the plant PM and cell wall. Plant actin dynamics are essential to this response, but it remains unclear how exocytosis and the cytoskeleton are linked in space and time to form functional CWAs. Here, we show that actin-dependent trafficking to immune response sites of Arabidopsis thaliana delivers membrane-integrated FORMIN4, which in turn contributes to local cytoskeletal dynamics. Total internal reflection fluorescence (TIRF) microscopy combined with controlled induction of FORMIN4-GFP expression reveals a dynamic population of vesicular bodies that accumulate to form clusters at the PM through an actin-dependent process. Deactivation of FORMIN4 and its close homologs partially compromises subsequent defense and alters filamentous actin (F-actin) distribution at mature CWAs. The localization of FORMIN4 is stable and segregated from the dynamic traffic of the endosomal network. Moreover, the tessellation of FORMIN4 at the PM with meso-domains of PEN3 reveals a fine spatial segregation of destinations for actin-dependent immunity cargo. Together, our data suggest a model where FORMIN4 is a spatial feedback element in a multi-layered, temporally defined sequence of cytoskeletal response. This positional feedback makes a significant contribution to the distribution of actin filaments at the dynamic CWA boundary and to the outcomes of pre-invasion defense.

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

Copper is an essential micronutrient but has toxic effects at high concentrations. Bryophytes are remarkably tolerant to elevated levels of copper but we wondered if this tolerance might be species dependent. Therefore, in three moss species, Physcomitrella patens, Mielichhoferia elongate and Pohlia drummondii, the accumulation of copper was compared with semiquantitative SEM-EDX analyses after six weeks of cultivation on copper containing media. We investigated the role of the copper-linked anion and applied copper as CuCl2, CuSO4 and CuEDTA, respectively. Line scans along the growth axis of moss gametophores allowed for a detailed analysis of copper detection from the base towards the tip. Mosses originating from metal-containing habitats (i.e. M. elongate and P. drummondii) revealed a lower accumulation of copper when compared to the non-adapted P. patens. CuEDTA had a shielding effect in all three species and copper levels differed greatly from CuCl2 or CuSO4. The detection of reactive oxygen species (ROS), H2O2 and O-2(-), was further used to indicate stress levels in the gametophore stems. ROS staining was increased along the whole stem and the tip in the non-adapted species P. patens whereas the tolerant species M. elongate and P. drummondii generally showed less staining located mainly at the base of the stem. We discuss the relation between metal accumulation and ROS production using indicator dyes in the three moss species. As moss gametophores are very delicate structures, ROS staining provide an excellent alternative to spectrophotometric analyses to estimate stress levels. (C) 2016 Elsevier Masson SAS. All rights reserved.

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.

•Metal stress caused a decreased ratio of gametophyte to protonema.•The anion crucially influenced tolerance and plant development.•Tolerance of P. patens towards added metals: Cl−=SO42−<EDTA4−.•The reduction of growth is best explained by free metal cation concentrations.•Phosphor rich nutrient media immobilize most metal salts.

Bryophytes are well-studied model systems for the investigation of uptake mechanisms and pollutant tolerance of plants. The moss Physcomitrella patens is among the most intensively studied organisms with regard to physiological and cellular pathways; however, direct ecological studies on metal stress are rare in this species. We conducted growth experiments on copper and zinc containing media for five and ten weeks, respectively in order to investigate the element and anion dependent metal uptake by energy-dispersive X-ray spectroscopy on a scanning electron microscope. We compared the uptake of metals to the growth and morphology of leaves and protonemata of P. patens. The metal content of both tissues was strongly correlated with availability of free metal cations. Differences between copper and zinc uptake included the limitation of zinc uptake found after five weeks for both tissues. No further sequestration of metal occurred after week five. The content of up to 1.3 wt% zinc and 0.4 wt% copper in the dry biomass correlated negatively to leafy gametophyte growth and to cell length in both tissues. No such correlation to protonema growth or cell width was found. The shortening of protonema cells exposed to zinc did not necessarily lead to decreased protonemal growth. This indicates a compensation of the shorter cells by increased cell division of the protonema filaments. (C) 2015 Elsevier B.V. All rights reserved.

Biochar production and subsequent soil incorporation could provide carbon farming solutions to global climate change and escalating food demand. There is evidence that biochar amendment causes fundamental changes in soil nutrient cycles, often resulting in marked increases in crop production, particularly in acidic and in infertile soils with low soil organic matter contents, although comparable outcomes in temperate soils are variable. We offer insight into the mechanisms underlying these findings by focusing attention on the soil nitrogen (N) cycle, specifically on hitherto unmeasured processes of organic N cycling in arable soils. We here investigated the impacts of biochar addition on soil organic and inorganic N pools and on gross transformation rates of both pools in a biochar field trial on arable land (Chernozem) in Traismauer, Lower Austria. We found that biochar increased total soil organic carbon but decreased the extractable organic C pool and soil nitrate. While gross rates of organic N transformation processes were reduced by 50-80%, gross N mineralization of organic N was not affected. In contrast, biochar promoted soil ammonia-oxidizer populations (bacterial and archaeal nitrifiers) and accelerated gross nitrification rates more than two-fold. Our findings indicate a de-coupling of the soil organic and inorganic N cycles, with a build-up of organic N, and deceleration of inorganic N release from this pool. The results therefore suggest that addition of inorganic fertilizer-N in combination with biochar could compensate for the reduction in organic N mineralization, with plants and microbes drawing on fertilizer-N for growth, in turn fuelling the belowground build-up of organic N. We conclude that combined addition of biochar with fertilizer-N may increase soil organic N in turn enhancing soil carbon sequestration and thereby could play a fundamental role in future soil management strategies.

Email of the presenting author: wolfram.adlassnig@univie.ac.at Biofilms are of crucial importance for the remediation of contaminated waters. Cyanobacteria of the genus Phormidium form dense mats in alpine creeks. The bacterial filaments are covered by a gelatinous sheath that adsorbs a variety of elements. In a copper contaminated creek in the Austrian Alps, an extensive, Phormidium dominated biofilm accumulates 3.9 ± 1.8% copper, thereby completely remediating the water of the creek by immobilising the copper. This study investigates the structure and ultrastructure of the biofilm with special regard to the localisation and speciation of the copper. The biofilm was chemically fixed or plunge frozen; further preparation included cryo-substition with ultrathin sectioning as well as freeze drying, conventional sectioning and analysis of whole mounts. Microscopic techniques comprised confocal microscopy, polarised and phase contrast light microscopy, X-ray microanalysis as well as scanning and transmission electron microscopy. The biofilm exhibits an extraordinary thickness of up to 22 cm with only the top layer containing living cells. It consists almost exclusively of filamentous Phormidium growing in clearly distinct layers (Fig. 1). Other bacteria like Bacillus are restricted to the surface of the biofilm. The copper is not evenly distributed in the biofilm but occurs as distinct crystals, probably consisting of the secondary copper mineral Sampleite [NaCaCu5(PO4)4 ∙ H2O] with a diameter of about 10 μm between the bacterial filaments (Fig. 2). Furthermore, copper is also found in the sheaths of the bacteria. Here, transmission electron microscopy suggests that this copper is not simply adsorbed but occurs in abundant submicroscopic electron-dense particles at the surface of the sheaths. These results show that the process of copper immobilisation by Phormidum biofilms is far more complex than simple passive adsorption and includes processes of biomineralisation. The speciation of the copper as mineral particles within the biofilm indicates that the immobilisation of metals is permanent which is confirmed by analysis of old, subfossile layers of the biofilm. These findings do not only enlighten the mechanism of metal immobilisation by Phormidium but also encourage its use for bioremediation of mine waste waters.

Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.

Using iron-deprived (-Fe) chlorotic as well as green iron-deficient (51 mu M Fe) and iron-sufficient supplied (50 mu M Fe) leaves of young hydroponically reared Brassica napus plants, we explored iron deficiency effects on triggering programmed cell death (PCD) phenomena. Iron deficiency increased superoxide anion but decreased hydroxyl radical (center dot OH) formation (TBARS levels). Impaired photosystem II efficiency led to hydrogen peroxide accumulation in chloroplasts; NADPH oxidase activity, however, remained on the same level in all treatments. Non-autolytic PCD was observed especially in the chlorotic leaf of iron-deprived plants, to a lesser extent in iron-deficient plants. It correlated with higher DNAse-, alkaline protease- and caspase-3-like activities, DNA fragmentation and chromatin condensation, hydrogen peroxide accumulation and higher superoxide dismutase activity. A significant decrease in catalase activity together with rising levels of dehydroascorbic acid indicated a strong disturbance of the redox homeostasis, which, however, was not caused by center dot OH formation in concordance with the fact that iron is required to catalyse the Fenton reaction leading to center dot OH generation. This study documents the chain of events that contributes to the development of non-autolytic PCD in advanced stages of iron deficiency in B. napus leaves. (C) 2013 Elsevier B.V. All rights reserved.

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.

Mining for heavy metals usually has a strong impact on the environment, including the formation of spoil heaps, mine tailings and mine drainage, all of which are heavily contaminated. Heavy metals are generally regarded as toxic for most organisms, including amphibians, although the effects of heavy metals may be extremely complex and sometimes even positive. This study presents a survey of observations of amphibians in habitats severely contaminated by mining for heavy metals in Central and Eastern Europe. Rocky spoil heaps and sandy mine tailings were generally found to be devoid of amphibians. In moist habitats, especially streams, puddles and ponds fed by drainage water, however, six species of amphibians were observed, i.e., Bombina variegata, Rana ridibunda, R. temporaria, Bufo viridis, Salamandra salamandra and S. atra. All six species were found in habitats superficially similar to their typically preferred habitats, e. g., Bombina variegata in small puddles, Salamandra salamandra larvae in a swiftly running stream. Moderately increased concentrations of copper, arsenic, antimony and other elements and an acidic pH of soil and water did not keep amphibians away. Highly contaminated or extremely acidic water bodies are usually devoid of amphibians even though they may be present in the surroundings, suggesting that amphibians may be capable of recognising and avoiding extreme degrees of contamination. With the uptake of the pollutants being highly probable, some amphibians appear to possess a limited tolerance against heavy metals.

The Schwarzwand is a unique hygric, Cu-contaminated habitat formed by mining activities from the 16th to 18th century. Today, a large spoil heap and several creeks fed by Cu-rich mine drainage are present. The vegetation of the Schwarzwand differs clearly from the surrounding subalpine forests. It is by no means impoverished but rather a hotspot of biodiversity. Interestingly, most of the Cu precipitates within the Schwarzwand and the creeks leave the Schwarzwand virtually clean. This study maps the distribution of Cu within the Schwarzwand and within selected vascular plants, moss and microorganisms and correlates them with water and soil chemistry in order to identify the sinks of Cu and to elucidate the remarkable capability of the Schwarzwand for natural attenuation.Two types of water could be distinguished, one acidic precipitating limonite with a constant Cu content of about 0.6 mg L (1), and one circumneutral, which decreases far more rapidly in Cu content than would be expected due to chemical considerations. A dense microbial mat covering most of the bed of the circumneutral creeks could be identified as the main sink. It consists of the cyanobacterium Phormidium sp. and retains Cu both by adsorption to mucilaginous sheaths and by precipitation as secondary minerals such as sampleite. Layers of dead biofilm can be found covered by a few centimetres of soil at the banks of the circumneutral creeks; the extremely high concentration and the low solubility of Cu in this soil indicates permanent immobilisation of the metals. High concentrations of Cu were also found in mosses of the family Bryaceae which, however, play a negligible role for the metal retention of the habitat due to their low biomass.The retention of Cu within the Schwarzwand is a remarkable example of the sustainable self-cleaning of a contaminated habitat which takes place without any human intervention. The artificial establishment of microbial communities similar to the Schwarzwand could result in cheap and sustainable strategies for the remediation of suitable metal-contaminated waters. (C) 2013 Elsevier Ltd. All rights reserved.

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.