Confocal Fluorescence Ratio Imaging of Ion Activities in Plant Cells

Fluorescent probes allow measurement of dynamic changes of calcium and pH in living cells. Imaging using confocal scanning laser microscopy provides a route to spatially map these dynamics over time in single optical sections or in 3-D images. We have developed a dual-excitation confocal system to allow ratio measurements of pH and calcium, that compensate for changes in dye distribution, leakage and photobleaching. Application of these techniques to plant tissues is complicated by the difficulty in loading the tissues with dye. We describe a new technique to assist dye loading in intact leaves of Lemna using a pre-treatment with cutinase. Once within plant tissues, many dyes compartmentalise into the vacuole. We report the use of chloromethylfluorescein diacetate as an alternative to BCECF [2\u27 ,7\u27-bis-(2-carboxyethyl)-5-(and 6)carboxyfluorescein] as a pH probe with greater cytoplasmic retention times. In addition, the confocal system allowed discrimination of signals from different compartments and permitted simultaneous measurement of vacuolar and cytoplasmic pH ratios in epidermal strips from Hordeum. We have developed a series of software tools to extract quantitative data from multi-dimensional images and illustrate these approaches with reference to pollen tube growth in Lilium and peptide-evoked changes in pH and calcium in stomata! guard cells from Commelina and Vicia

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement

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

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 photoreceptor(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

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 photoreceptor(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

Continuous imaging of amino-acid translocation in intact mycelia of Phanerochaete velutina reveals rapid, pulsatile fluxes

Nitrogen translocation by woodland fungi is ecologically important, however, techniques to study long-distance amino-acid transport in mycelia currently have limited spatial and temporal resolution. We report a new continuous, noninvasive imaging technique for β-emitters that operates with submillimetre spatial resolution and a practical sampling interval of 10-60 min. • Transport of the nonmetabolized, 14C-labelled amino-acid analogue, α-aminoisobutyric acid (AIB) was imaged using a photon-counting camera as it was transported in foraging mycelium of the cord-forming woodland fungus, Phanerochaete velutina, grown over an intensifying screen in microcosms. • The maximum acropetal transport velocity of 14C-AIB to the colony margin was 50 mm h-1 (average 23 mm h-1), with a mass transfer of 4.6-51.5 pmol 14C-AIB h-1 per cord. Transport in cords had a pulsatile component with a period of 11-12 h. • Transport was significantly faster than diffusion, consistent with rapid cycling of nutrients throughout the mycelium between loading and sink regions. The increased spatial and temporal resolution of this method also revealed the rhythmic nature of transport in this fungus for the first time. © New Phytologist (2002)

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

Noncircadian oscillations in amino acid transport have complementary profiles in assimilatory and foraging hyphae of Phanerochaete velutina

• Cord-forming woodland basidiomycete fungi form extensive, interconnected mycelial networks that scavenge nitrogen (N) efficiently. We have developed techniques to study N dynamics in such complex mycelial systems in vivo. • Uptake and distribution of the nonmetabolised, 14C-labelled amino-acid analogue, α-aminoisobutyrate (14C-AIB) was continuously imaged in Phanerochaete velutina growing across scintillation screens using an enhanced photon-counting camera. • Oscillations in the 14C-AIB signal were observed for both the assimilatory hyphae in the inoculum and the foraging hyphae, but with complementary profiles. Pulses were asymmetric, with an abrupt switch between each exponential decay phase and the next rising phase. The period of the oscillations was 16 h at 21°C, but showed a strong temperature dependence with a temperature coefficient of 2.1. Oscillations occurred in the absence of obvious pulses in growth. • Some, but not all, of the features of the oscillations were simulated using a model of amino acid accumulation and transport that included both vacuolar uptake, and release once an intravacuolar concentration threshold was exceeded. The combination of imaging and modelling provides a useful framework to understand N fluxes in vivo

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