Network traits predict ecological strategies in fungi.

Colonization of terrestrial environments by filamentous fungi relies on their ability to form networks that can forage for and connect resource patches. Despite the importance of these networks, ecologists rarely consider network features as functional traits because their measurement and interpretation are conceptually and methodologically difficult. To address these challenges, we have developed a pipeline to translate images of fungal mycelia, from both micro- and macro-scales, to weighted network graphs that capture ecologically relevant fungal behaviour. We focus on four properties that we hypothesize determine how fungi forage for resources, specifically: connectivity; relative construction cost; transport efficiency; and robustness against attack by fungivores. Constrained ordination and Pareto front analysis of these traits revealed that foraging strategies can be distinguished predominantly along a gradient of connectivity for micro- and macro-scale mycelial networks that is reminiscent of the qualitative 'phalanx' and 'guerilla' descriptors previously proposed in the literature. At one extreme are species with many inter-connections that increase the paths for multidirectional transport and robustness to damage, but with a high construction cost; at the other extreme are species with an opposite phenotype. Thus, we propose this approach represents a significant advance in quantifying ecological strategies for fungi using network information.

Foraging by a wood-decomposing fungus is ecologically adaptive.

We show that fungi that forage for wood do not conform to the paradigm of symmetric radial growth and grow asymmetrically by default. Asymmetry is further accentuated by contact with a resource that also partially polarizes growth in the direction of the resource. Despite marked changes at the perimeter, overall growth allocation on an area basis is, however, unchanged implying sophisticated regulation at the colony level. Using mathematical models, we show that this behaviour is best explained as a local response of the immediate segment contacting the resource. The model reveals that foraging behaviour is adaptive but only for resources that are clustered in space and is selectively neutral for randomly scattered resources. This clustered spatial distribution matches that found in the natural environment. Modelling also shows that the foraging strategy used by these fungi involves substantial risks as well as benefits.

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum.

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.

Confocal imaging of glutathione redox potential in living plant cells.

Reduction-oxidation-sensitive green fluorescent protein (roGFP1 and roGFP2) were expressed in different sub-cellular compartments of Arabidopsis and tobacco leaves to empirically determine their performance as ratiometric redox sensors for confocal imaging in planta. A lower redox-dependent change in fluorescence in combination with reduced excitation efficiency at 488 nm resulted in a significantly lower dynamic range of roGFP1 than for roGFP2. Nevertheless, when targeted to the cytosol and mitochondria of Arabidopsis leaves both roGFPs consistently indicated redox potentials of about -320 mV in the cytosol and -360 mV in the mitochondria after pH correction for the more alkaline matrix pH. Ratio measurements were consistent throughout the epidermal cell layer, but results might be attenuated deeper within the leaf tissue. Specific interaction of both roGFPs with glutaredoxin in vitro strongly suggests that in situ both variants preferentially act as sensors for the glutathione redox potential. roGFP2 targeted to plastids and peroxisomes in epidermal cells of tobacco leaves was slightly less reduced than in other plasmatic compartments, but still indicated a highly reduced glutathione pool. The only oxidizing compartment was the lumen of the endoplasmic reticulum, in which roGFP2 was almost completely oxidized. In all compartments tested, roGFP2 reversibly responded to perfusion with H(2)O(2) and DTT, further emphasizing that roGFP2 is a reliable probe for dynamic redox imaging in planta. Reliability of roGFP1 measurements might be obscured though in extended time courses as it was observed that intense irradiation of roGFP1 at 405 nm can lead to progressive photoisomerization and thus a redox-independent change of fluorescence excitation ratios.

Imaging complex nutrient dynamics in mycelial networks.

Transport networks are vital components of multi-cellular organisms, distributing nutrients and removing waste products. Animal cardiovascular and respiratory systems, and plant vasculature, are branching trees whose architecture is thought to determine universal scaling laws in these organisms. In contrast, the transport systems of many multi-cellular fungi do not fit into this conceptual framework, as they have evolved to explore a patchy environment in search of new resources, rather than ramify through a three-dimensional organism. These fungi grow as a foraging mycelium, formed by the branching and fusion of threadlike hyphae, that gives rise to a complex network. To function efficiently, the mycelial network must both transport nutrients between spatially separated source and sink regions and also maintain its integrity in the face of continuous attack by mycophagous insects or random damage. Here we review the development of novel imaging approaches and software tools that we have used to characterise nutrient transport and network formation in foraging mycelia over a range of spatial scales. On a millimetre scale, we have used a combination of time-lapse confocal imaging and fluorescence recovery after photobleaching to quantify the rate of diffusive transport through the unique vacuole system in individual hyphae. These data then form the basis of a simulation model to predict the impact of such diffusion-based movement on a scale of several millimetres. On a centimetre scale, we have used novel photon-counting scintillation imaging techniques to visualize radiolabel movement in small microcosms. This approach has revealed novel N-transport phenomena, including rapid, preferential N-resource allocation to C-rich sinks, induction of simultaneous bi-directional transport, abrupt switching between different pre-existing transport routes, and a strong pulsatile component to transport in some species. Analysis of the pulsatile transport component using Fourier techniques shows that as the colony forms, it self-organizes into well demarcated domains that are identifiable by differences in the phase relationship of the pulses. On the centimetre to metre scale, we have begun to use techniques borrowed from graph theory to characterize the development and dynamics of the network, and used these abstracted network models to predict the transport characteristics, resilience, and cost of the network.

Quantifying dynamic resource allocation illuminates foraging strategy in Phanerochaete velutina.

Saprotrophic woodland fungi forage for mineral nutrients and woody resources by extension of a mycelial network across the forest floor. Different species explore at different rates and establish networks with qualitatively differing architecture. However, detailed understanding of fungal foraging behaviour has been hampered by the absence of tools to quantify resource allocation and growth accurately and non-invasively. To solve this problem, we have used photon-counting scintillation imaging (PCSI) to map and quantify nutrient allocation and localised growth simultaneously in heterogeneous resource environments. We show that colonies spontaneously shift to an asymmetric growth pattern, even in the absence of added resources, often with a distinct transition between the two growth phases. However, the extent of polarisation was much more pronounced and focussed in the presence of an additional cellulose resource. In this case, there was highly localised growth, often at the expense of growth elsewhere in the colony, and marked accumulation of (14)C-AIB in the sector of the colony with the added resource. The magnitude of the response was greatest when resource was added around the time of the endogenous developmental transition. The focussed response required a metabolisable resource, as only limited changes were seen with glass fibre discs used to mimic the osmotic and thigmotropic stimuli upon resource addition. Overall the behaviour is consistent with an adaptive foraging strategy, both to exploit new resources and also to redirect subsequent foraging effort to this region, presumably with an expectation that the probability of finding additional resources is increased.

Emergence of self-organised oscillatory domains in fungal mycelia.

Fungi play a central role in the nutrient cycles of boreal and temperate forests. In these biomes, the saprotrophic wood-decay fungi are the only organisms that can completely decompose woody plant litter. In particular, cord-forming basidiomycete fungi form extensive mycelial networks that scavenge scarce mineral nutrients and translocate them over long distances to exploit new food resources. Despite the importance of resource allocation, there is limited information on nutrient dynamics in these networks, particularly for nitrogen, as there is no suitable radioisotope available. We have mapped N-translocation using photon-counting scintillation imaging of the non-metabolised amino acid analogue, (14)C-aminoisobutyrate. We describe a number of novel phenomena, including rapid, preferential N-resource allocation to C-rich sinks, induction of simultaneous bi-directional N-transport, abrupt switching between different pre-existing transport routes, and emergence of locally synchronised, oscillatory phase domains. It is possible that such self-organised oscillatory behaviour is a mechanism to achieve global co-ordination in the mycelium.

Fourier-based spatial mapping of oscillatory phenomena in fungi.

Microorganisms display a range of oscillatory phenomena that operate over different temporal scales. Fourier analysis provides a compact description of such oscillations in terms of their frequency, magnitude and phase. However, in the majority of studies there is no explicit consideration of the spatial organisation of the oscillation. Here we describe procedures and a software package to map oscillatory phenomena in microorganisms in both the time and frequency domains. Key parameters of interest, such as frequency, phase or magnitude of the oscillations, are presented as pseudo-colour coded maps. This maintains the spatial information in the image and greatly facilitates understanding of potentially complex propagating waves or development of oscillatory domains with distinct behaviour. We illustrate the utility of this system with reference to spatial analysis of the pulsatile component to amino acid transport in mycelial systems of Phanerochaete velutina and Coniophora puteana, and actin-myosin based contractions in Physarum polycephalum.

Nitrite reduces cytoplasmic acidosis under anoxia.

The ameliorating effect of nitrate on the acidification of the cytoplasm during short-term anoxia was investigated in maize (Zea mays) root segments. Seedlings were grown in the presence or absence of nitrate, and changes in the cytoplasmic and vacuolar pH in response to the imposition of anoxia were measured by in vivo (31)P nuclear magnetic resonance spectroscopy. Soluble ions and metabolites released to the suspending medium by the anoxic root segments were measured by high-performance liquid chromatography and (1)H nuclear magnetic resonance spectroscopy, and volatile metabolites were measured by gas chromatography and gas chromatography-mass spectrometry. The beneficial effect of nitrate on cytoplasmic pH regulation under anoxia occurred despite limited metabolism of nitrate under anoxia, and modest effects on the ions and metabolites, including fermentation end products, released from the anoxic root segments. Interestingly, exposing roots grown and treated in the absence of nitrate to micromolar levels of nitrite during anoxia had a beneficial effect on the cytoplasmic pH that was comparable to the effect observed for roots grown and treated in the presence of nitrate. It is argued that nitrate itself is not directly responsible for improved pH regulation under anoxia, contrary to the usual assumption, and that nitrite rather than nitrate should be the focus for further work on the beneficial effect of nitrate on flooding tolerance.

The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi.

Mycelial fungi have a growth form which is unique among multicellular organisms. The data presented here suggest that they have developed a unique solution to internal solute translocation involving a complex, extended vacuole. In all filamentous fungi examined, this extended vacuole forms an interconnected network, dynamically linked by tubules, which has been hypothesized to act as an internal distribution system. We have tested this hypothesis directly by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker. Predictive simulation models were then used to determine the transport characteristics over extended length scales. This modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimeters to centimeters. Flux through the pathway is regulated by the dynamic tubular connections involving homotypic fusion and fission. There is also a strongly predicted interaction among vacuolar organization, predicted diffusion transport distances, and the architecture of the branching colony margin.

Cell-specific measurement of cytosolic glutathione in poplar leaves.

The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves or roots; however, there is little information on the effect that such asymmetries have on the actual GSH concentration in each compartment or cell type. In the present study in situ labelling with monochlorobimane (MCB) in combination with confocal laser scanning microscopy was used to quantify GSH in each of the main cell types of poplar leaves from fluorescence of the GSB conjugate formed. Comparison of results from the in situ approach with total GSH levels measured in vitro by high-performance liquid chromatography suggested that only the cytosolic GSH pool was labelled using this approach. This suggests that an appropriate GST was not present within the chloroplasts to catalyse the conjugation reaction and that chloroplastic GSH does not rapidly exchange with the cytoplasmic pool under the conditions of the assay. Cytosolic GSH levels were between 0.2 and 0.3 mm for both photosynthetic and non-photosynthetic (epidermal) cell types in wild-type poplar leaves. Cytosolic levels increased by around two-fold in transgenic poplars over-expressing bacterial gamma-glutamylsynthetase (gamma-ECS) in the cytosol of all cell types, but there was no concomitant increase in the chloroplastic GSH pool.

Confocal imaging of metabolism in vivo: pitfalls and possibilities.

Confocal laser scanning microscopy (CLSM) has had wide application in morphological studies and ion imaging in plants, but little impact so far on biochemical investigations. This position is likely to change as the range of fluorescent probes increases. To illustrate the type of kinetic information that can be obtained using CLSM in an intact, living system, an analysis has been made of the two-step detoxification of monochlorobimane (MCB) following conjugation to glutathione (GSH) by a glutathione S-transferase in the cytoplasm and vacuolar sequestration of the fluorescent glutathione S-bimane (GSB) by a glutathione S-conjugate (GSX) pump. Fluorescence from the cytoplasm and vacuole of individual trichoblasts and atrichoblasts was measured from time-series of (x, y) optical sections in the elongation zone of Arabidopsis root tips. Intensity changes were calibrated and converted to amounts using compartment volumes, measured by stereological techniques. The data were well described using pseudo-first-order kinetics for the conjugation reaction and either Michaelis-Menten kinetics (Model I), or, as the GSX-pump was operating close to V(max), a pseudo-zero-order reaction (Model II), for the GSX-pump. Analysis of 15 individual cells from two roots gave [GSH](cyt) in the range 1.8-4 mM. GST activity was relatively constant on a cell basis in one root, but increased markedly in the other, giving a net increase in conjugation activity as cells progressed through the elongation zone. In contrast, GSX-pump activity increased in parallel with the increase in cell size in both roots, effectively maintaining a constant transport activity per unit root length or estimated vacuole surface area.

Measurement of glutathione levels in intact roots of Arabidopsis.

Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S-bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15-20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S-conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2-3 mM in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell-cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.

Direct measurement of glutathione in epidermal cells of intact Arabidopsis roots by two-photon laser scanning microscopy.

Two-photon laser scanning microscopy (TPLSM) was used to directly measure glutathione (GSH) as its fluorescent glutathione S-bimane conjugate (GSB) in developing root hair cells (trichoblasts) and non-root hair cells (atrichoblasts) of intact Arabidopsis roots. In comparison to confocal microscopy, TPLSM showed more detail deep within the tissue with less signal attenuation. The total level of GSB labelling reached a plateau after 60 min in both trichoblasts and atrichoblasts, reflecting depletion of GSH. GSB was formed initially in the cytoplasm and was subsequently transported into the vacuole. The volume ratio of vacuole to cytoplasm was determined using the Cavalieri estimator of volume and used to calculate the amount of GSB per volume of cytoplasm in each cell type. At the end of the time-course the cytoplasmic concentration of GSB was 2.7 +/- 0.5 mM (n = 5) in trichoblasts and 5.5 +/- 0.8 mM (n = 5) in atrichoblasts. In trichoblasts this value represents the initial concentration of GSH in the cytoplasm. Labelling of roots with monochlorobimane (MCB) on ice led to the formation of GSB in the cytoplasm, but prevented vacuolar sequestration. After washing prelabelled roots and transfer to room temperature, vacuolar transport resumed. Although no free MCB was present the total amount of GSB in atrichoblasts increased further, indicating that the higher values recorded in the atrichoblasts might reflect additional symplastic transport and sequestration of GSB from neighbouring cells.

The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin.

In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided. The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin. Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function. We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ). In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4. A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus. In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes. We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)). Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle.

pH gradients are not associated with tip growth in pollen tubes of Lilium longiflorum.

The cytoplasmic pH of growing pollen tubes of Lilium longiflorum Thunb. was measured using the pH-sensitive fluorescent dye 2',7'-bis-(carboxyethyl)-5(6')-carboxyfl uorescein and confocal fluorescence ratio imaging. The average cytoplasmic pH in the clear zone of the pollen tube tip was pH 7.11, and no consistent pH gradients were detected in the clear zone, averaging around -1.00 milli pH unit microm(-1), or along the first 50 microm of the tube (3.62 milli pH units microm[-1]). In addition, no correlation was observed between the absolute tip cytoplasmic pH or the pH gradient and the pollen tube growth rates. Shifts of external pH to more acidic pH values (pH 4.5) caused a relatively small acidification by 0.18 pH units, whereas a more alkaline external pH >7.0 caused a dramatic increase in cytoplasmic pH and growth stopped immediately. Stimulation of the plasma membrane H+-ATPase by fusicoccin, resulted in an increase of tube growth but no change in cytoplasmic pH. On the other hand, vanadate (250-500 microM), a putative inhibitor of the pump, stopped tube growth and a slight cytoplasmic alkalinisation of 0.1 pH units was observed. Vanadate also arrested fusicoccin-stimulated growth and stimulated an increased alkalinisation of around 0.2 pH units. External application of CaCl2 (10 mM) caused a small acidification of less than 0.1 pH units in the clear zone, whilst LaCl3 (250 microM) caused slight and rather variable perturbations in cytoplasmic pH of no more than 0.1 pH units. Both treatments stopped growth. It was inferred from these data that tip-acid cytoplasmic pH gradients do not play a central role in the organisation or maintenance of pollen tube tip growth.

Four-dimensional imaging of living chondrocytes in cartilage using confocal microscopy: a pragmatic approach.

Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.

Genomic sequence of a calnexin homolog from Arabidopsis thaliana.

Calnexin is a membrane-bound protein of the ER in animal cells (Wada et al., 1991). It shows considerable similarity to the major calcium-sequestering protein of the ER lumen, calreticulin, with two calcium-binding regions--a high-affinity, low-capacity region in the ER lumen and a low-affinity, high-capacity region in the cytoplasm. The protein is postulated to act as a calcium-regulated chaperone during protein maturation (Ou et al., 1993). We have isolated a genomic sequence showing significant homology to the animal gene over the predicted coding sequence (Table I). A partial cDNA from Zea mays was isolated from an expression library made from 6-d coleoptiles (Clontech, Palo Alto, CA). The library was screened using a monoclonal antibody raised against a small number of microsomal proteins resulting from a partial purification of plasma membrane Ca2+ ATPase (Briars et al., 1988). The partial cDNA showed sequence homology to the calcium-binding region common to calreticulin and calnexin. The fragment was used to screen a genomic library constructed from Arabidopsis thaliana (cv Larasbonerecta), and a 15-kb fragment was isolated and subcloned and the relevant subfragments were sequenced. The coding region contains five introns, two in the N-terminal region and three in the C-terminal region. The predicted amino acid sequence shows a high level of homology with the animal calnexin, although the terminal highly acidic calcium-binding region is shorter. A cDNA for a putative homolog of calnexin was isolated from A. thaliana (cv Columbia) by Huang et al.(1993); our coding sequence shows 85% identity and 92% similarity determined by FASTA (Wisconsin Genetics Computer Group package); however, the differences are greater than would be expected between cultivars of the same species. A Southern blot probed with DNA from the central calcium-binding region shows multiple bands. This, combined with the sequence heterogeneity, suggests that calnexin belongs to a family of related genes.

Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories.