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.

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.

Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in lemna trisulca L

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo-damage in strong blue light (SBL). The photo- receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue-light photoreceptor in chloroplast movements was investigated. Time-lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL-dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis-trans isomerization of xanthophylls, thus photo-isomerization may be a critical link in the signal transduction pathway.

The role of calcium in blue-light-dependent chloroplast movement in lemna trisulca L

Chloroplast movements are a normal physiological response to changes in light intensity and provide a good model system to analyse the signal transduction pathways following light perception. Blue-light-dependent chloroplast movements were observed in Lemna trisulca using confocal optical sectioning and 3-D reconstruction or photometric measurements of leaf transmission. Chloroplasts moved away from strong blue light (SBL) towards the anticlinal walls (profile position), and towards the periclinal walls (face position) under weak blue light (WBL) over about 20-40 min. Cytoplasmic calcium ([Ca2 + ]cyt) forms part of the signalling system in response to SBL as movements were associated with small increases in [Ca2 + ]cyt and were blocked by antagonists of calcium homeostasis, including EGTA, nifedipine, verapamil, caffeine, thapsigargin, TFP (trifluoperazine), W7 and compound 48/80. Treatments predicted to affect internal Ca2 + stores gave the most rapid and pronounced effects. In addition, artificially increasing [Ca2 + ]cyt in darkness using the Ca2 + ionophore A23187 and high external Ca2 + (or Sr2 + ), triggered partial movement of chloroplasts to profile position analogous to a SBL response. These data are all consistent with [Ca2 + ]cyt acting as a signal in SBL responses; however, the situation is more complex given that both WBL and SBL responses were inhibited to a similar extent by all the Ca2 + -signalling antagonists used. As the direction of chloroplast movement in WBL is exactly opposite to that in SBL, we conclude that, whilst proper regulation of [Ca2 + ]cyt homeostasis is critical for both SBL and WBL responses, additional factors may be required to specify the direction of chloroplast movement.