Plant and fungal gene expression coupled with stable isotope labeling provide novel information on sulfur uptake and metabolism in orchid mycorrhizal protocorms.

Orchid mycorrhiza (OM) represents an unusual symbiosis between plants and fungi because in all orchid species carbon is provided to the host plant by the mycorrhizal fungus at least during the early stages of orchid development, named a protocorm. In addition to carbon, orchid mycorrhizal fungi provide the host plant with essential nutrients such as phosphorus and nitrogen. In mycorrhizal protocorms, nutrients transfer occurs in plant cells colonized by the intracellular fungal coils, or pelotons. Whereas the transfer of these vital nutrients to the orchid protocorm in the OM symbiosis has been already investigated, there is currently no information on the transfer of sulfur (S). Here, we used ultra-high spatial resolution secondary ion mass spectrometry (SIMS) as well as targeted gene expression studies and laser microdissection to decipher S metabolism and transfer in the model system formed by the Mediterranean orchid Serapias vomeracea and the mycorrhizal fungus Tulasnella calospora. We revealed that the fungal partner is actively involved in S supply to the host plant, and expression of plant and fungal genes involved in S uptake and metabolism, both in the symbiotic and asymbiotic partners, suggest that S transfer most likely occurs as reduced organic forms. Thus, this study provides original information about the regulation of S metabolism in OM protocorms, adding a piece of the puzzle on the nutritional framework in OM symbiosis.

Stable isotope cellular imaging reveals that both live and degenerating fungal pelotons transfer carbon and nitrogen to orchid protocorms.

The objective of this study was to elucidate the transfer of nutrient elements in orchid symbiotic protocorms at the cellular level by imaging of stable isotope tracers. We address the long-standing question of whether nutrients move by transport across the symbiotic interface or solely by lysis of fungal pelotons. [U-(13) C]glucose and (15) NH4 (15) NO3 were added to Ceratobasidium sp. hyphae extending from symbiotic protocorms of Spiranthes sinensis. Isotope images were taken from resin-embedded sections of protocorms using ultra-high spatial resolution secondary ion mass spectrometry (SIMS). Analyses of regions of interest were conducted on isotope ratio images for fungal and host structures. Amyloplasts adjacent to young pelotons showed elevated (13) C/(12) C, which indicated that fungal carbon (C) was transferred from live hyphae. Senescent pelotons and their surrounding host cytoplasm showed significantly higher isotope ratios than young pelotons and surrounding host cytoplasm. These results indicate an inflow of C to senescent hyphae, which was then transferred to the host. The findings of this study provide some support for each of the two contradictory hypotheses concerning nutrient exchange in the symbiotic protocorm: the interface between the symbionts is involved before fungal senescence, and peloton degradation also releases a significant amount of C and nitrogen to host cells.

Allocation of Carbon from an Arbuscular Mycorrhizal Fungus, Gigaspora margarita, to Its Gram-Negative and Positive Endobacteria Revealed by High-Resolution Secondary Ion Mass Spectrometry.

Arbuscular mycorrhizal fungi are obligate symbionts of land plants; furthermore, some of the species harbor endobacteria. Although the molecular approach increased our knowledge of the diversity and origin of the endosymbiosis and its metabolic possibilities, experiments to address the functions of the fungal host have been limited. In this study, a C flow of the fungus to the bacteria was investigated. Onion seedlings colonized with Gigaspora margarita, possessing Candidatus Glomeribacter gigasporarum (CaGg, Gram-negative, resides in vacuole) and Candidatus Moeniiplasma glomeromycotorum (CaMg, Gram-positive, resides in the cytoplasm,) were labelled with 13CO2. The 13C localization within the mycorrhiza was analyzed using high-resolution secondary ion mass spectrometry (SIMS). Correlative TEM-SIMS analysis of the fungal cells revealed that the 13C/12C ratio of CaGg was the lowest among CaMg and mitochondria and was the highest in the cytoplasm. By contrast, the plant cells, mitochondria, plastids, and fungal cytoplasm, which are contributors to the host, showed significantly higher 13C enrichment than the host cytoplasm. The C allocation patterns implied that CaMg has a greater impact than CaGg on G. margarita, but both seemed to be less burdensome to the host fungus in terms of C cost.

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development.

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea. alpha-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and beta-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were alpha-1,3-glucan and chitosan, but after enzymatic digestion of alpha-1,3-glucan, beta-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed alpha-1,3-glucan and beta-1,3-glucan intermixed in the cell wall of infectious hyphae; however, alpha-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of alpha-1,3-glucan. Accumulation of alpha-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, alpha-1,3-glucan spatially and functionally masks beta-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea.

Evaluation of secondary ions related to plant tissue using least absolute shrinkage and selection operator.

With regard to life sciences, it is important to understand biological functions such as metabolic reactions at the cellular level. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) that can provide chemical mappings at 100 nm lateral resolutions is useful for obtaining three-dimensional maps of biological molecules in cells and tissues. TOF-SIMS spectra generally contain several hundred to several thousand secondary ion peaks that provide detailed chemical information. In order to manage such a large number of peaks, data analysis methods such as multivariate analysis techniques have been applied to TOF-SIMS data of complex samples. However, the interpretation of the data analysis results is sometimes still difficult, especially for biological samples. In this study, TOF-SIMS data of resin-embedded plant samples were analyzed using one of the sparse modeling methods, least absolute shrinkage and selection operator (LASSO), to directly select secondary ions related to biological structures such as cell walls and nuclei. The same sample was measured by optical microscopy and the same measurement area as TOF-SIMS was extracted in order to prepare a target image for LASSO. The same area of the TOF-SIMS and microscope data were fused to evaluate the influence of the image fusion on the TOF-SIMS spectrum information using principal component analysis. Specifically, the authors examined onion mycorrhizal root colonized with Gigaspora margarita (an arbuscular mycorrhizal fungus). The results showed that by employing this approach using LASSO, important secondary ions from biological samples were effectively selected and could be clearly distinguished from the embedding resin.

ATP-dependent but proton gradient-independent polyphosphate-synthesizing activity in extraradical hyphae of an arbuscular mycorrhizal fungus.

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A(1) or a protonophore, suggesting that ATP may not energize the reaction through H(+)-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.

Ultrastructure of rapidly frozen and freeze-substituted germ tubes of an arbuscular mycorrhizal fungus and localization of polyphosphate.

In arbuscular mycorrhizas (AM), the supply of phosphorus from the fungi is one of the most important benefits to the host plant. Here we describe for the first time the ultrastructure and polyphosphate (poly P) distribution in rapidly frozen and freeze-substituted germ tubes of the AM fungus Gigaspora margarita. At the ultrastructural level, phosphorus distribution was analysed using energy-filtering transmission electron microscopy, and poly P was detected using an enzyme-affinity method. Semithin sections and live cells were also stained with 4',6-diamidino-2-phenylindole, which is not specific but fluoresces yellow when viewed under UV irradiation by binding with poly P. The cryotechnique method showed that extensive elongate ellipsoid vacuoles containing a uniform electron-opaque material occupied most of the cell volume. Combining the results of multiple methods revealed that poly P was localized in a dispersed form in vacuoles and in the outer fungal cell wall. These results show the significant potential of AM fungi for phosphorus storage based on its localization in the extensive complement of vacuoles in thick hyphae. The mechanism of translocation of poly P in tubular vacuoles, and the role of poly P in the cell wall, need to be elucidated.

Uptake and Intraradical Immobilization of Cadmium by Arbuscular Mycorrhizal Fungi as Revealed by a Stable Isotope Tracer and Synchrotron Radiation µX-Ray Fluorescence Analysis.

Arbuscular mycorrhizal (AM) fungi can improve plant tolerance to heavy metal contamination. This detoxification ability may largely depend on how AM fungi influence the uptake and distribution of metals in host plants. Two experiments were performed in order to gain insights into the mechanisms underlying cadmium (Cd) tolerance in mycorrhizal plants. Stable isotope Cd106 and compartmented pots were adopted to quantify the contribution of the AM fungus, Rhizophagus irregularis, to the uptake of Cd by Lotus japonicus. Moreover, synchrotron radiation µX-ray fluorescence (SR-µXRF) was applied to localize Cd in the mycorrhizal roots at the sub-cellular level. The results obtained indicated that mycorrhizal colonization markedly enhanced Cd immobilization in plant roots. Less Cd was partitioned to plant shoots when only hyphae had access to Cd in the hyphal compartment than when roots also had direct access to the Cd pool. SR-µXRF imaging indicated that Cd absorbed by extraradical hyphae was translocated into intraradical fungal structures, in which arbuscules accumulated large amounts of Cd; however, plant cells without fungal structures and plant cell walls contained negligible amounts of Cd. The present results provide direct evidence for the intraradical immobilization of Cd absorbed by AM fungi, which may largely contribute to the enhanced tolerance of plants to Cd. Therefore, AM fungi may play a role in the phytostabilization of Cd-contaminated soil.

Cellular imaging of cadmium in resin sections of arbuscular mycorrhizas using synchrotron micro X-ray fluorescence.

Arbuscular mycorrhizal (AM) fungi function as extended roots and take an active part in plant acquisition of nutrients and also soil pollutants, such as heavy metals. The objective of this study was to establish a method to observe the localization of cadmium (Cd) Kα at subcellular levels using X-ray fluorescence (XRF) imaging with a synchrotron irradiation microbeam in resin-embedded sections of mycorrhizas. To evaluate the methodology, distributions of Cd in high-pressure-frozen Lotus japonicus-Rhizophagus irregularis mycorrhizal roots were compared between two treatments; Cd was exposed either to the roots or to the extraradical hyphae. Results showed that, in the latter treatment, Cd was restricted to fungal structures, whereas in the former, Cd was detected in cell walls of the two organisms. Plunge-frozen extraradical mycelium of Gigaspora margarita exposed to Cd showed high signals of Cd in the cell walls and vacuoles, and low in the cytoplasm. With selective staining and elemental mapping by electron-dispersive X-ray spectrometry (EDS), a positive correlation between distributions of Cd and P was revealed in the vacuole, which suggested polyP as a counter ion of Cd. These results indicated that there was no Cd relocation in rapidly frozen resin-embedded materials, therefore supporting the usefulness of this methodology.