Identification and interest of molecular markers to monitor plant Pi status.

BACKGROUND: Inorganic phosphate (Pi) is the sole source of phosphorus for plants. It is a limiting factor for plant yield in most soils worldwide. Due to economic and environmental constraints, the use of Pi fertilizer is and will be more and more limited. Unfortunately, evaluation of Pi bioavailability or Pi starvation traits remains a tedious task, which often does not inform us about the real Pi plant status. RESULTS: Here, we identified by transcriptomic studies carried out in the plant model Arabidopsis thaliana, early roots- or leaves-conserved molecular markers for Pi starvation, exhibiting fast response to modifications of phosphate nutritional status. We identified their homologues in three crops (wheat, rapeseed, and maize) and demonstrated that they offer a reliable opportunity to monitor the actual plant internal Pi status. They turn out to be very sensitive in the concentration range of 0-50 µM which is the most common case in the vast majority of soils and situations where Pi hardly accumulates in plants. Besides in vitro conditions, they could also be validated for plants growing in the greenhouse or in open field conditions. CONCLUSION: These markers provide valuable physiological tools for plant physiologists and breeders to assess phosphate bio-availability impact on plant growth in their studies. This also offers the opportunity to cope with the rising economical (shortage) and societal problems (pollution) resulting from the management of this critical natural resource.

Overexpression of the HcPT1.1 transporter in Hebeloma cylindrosporum alters the phosphorus accumulation of Pinus pinaster and the distribution of HcPT2 in ectomycorrhizae.

Ectomycorrhizal (ECM) fungi are associated with the roots of woody plants in temperate and boreal forests and help them to acquire water and nutrients, particularly phosphorus (P). However, the molecular mechanisms responsible for the transfer of P from the fungus to the plant in ectomycorrhizae are still poorly understood. In the model association between the ECM fungus Hebeloma cylindrosporum and its host plant Pinus pinaster, we have shown that the fungus, which possesses three H+:Pi symporters (HcPT1.1, HcPT1.2 and HcPT2), expresses mainly HcPT1.1 and HcPT2 in the extraradical and intraradical hyphae of ectomycorrhizae to transport P from the soil to colonized roots. The present study focuses on the role of the HcPT1.1 protein in plant P nutrition, in function of P availability. We artificially overexpressed this P transporter by fungal Agrotransformation and investigated the effect of the different lines, wild-type and transformed ones, on plant P accumulation, the distribution of HcPT1.1 and HcPT2 proteins in ectomycorrhizae by immunolocalization, and 32P efflux in an experimental system mimicking intraradical hyphae. Surprisingly, we showed that plants interacting with transgenic fungal lines overexpressing HcPT1.1 did not accumulate more P in their shoots than plants colonized with the control ones. Although the overexpression of HcPT1.1 did not affect the expression levels of the other two P transporters in pure cultures, it induced a strong reduction in HcPT2 proteins in ectomycorrhizae, particularly in intraradical hyphae, but still improved the P status of host plant shoots compared with non-mycorrhizal plants. Finally, 32P efflux from hyphae was higher in lines overexpressing HcPT1.1 than in the control ones. These results suggest that a tight regulation and/or a functional redundancy between the H+:Pi symporters of H. cylindrosporum might exist to ensure a sustainable P delivery to P. pinaster roots.

Improved rock phosphate dissolution from organic acids is driven by nitrate assimilation of bacteria isolated from nitrate and CaCO3-rich soil.

Until now, the solubilization capacities of insoluble mineral P by soil microorganisms have been screened in vitro with media containing NH4+ as a nitrogen source. This presence of NH4+ will lead to an acidification of the medium responsible for the solubilization of the insoluble P. However, besides proton release, the production of organic acids can play a very important role in the release of free P. This physiological mechanism can largely depend on the source of nitrogen (NH4+vs NO3-) assimilated by the bacteria but the influence of the N source on the production of organic acids has yet to be studied. Our aim was to investigate if the N source assimilated by bacteria and the soil characteristics such as the dominant N source (NH4+vs NO3-) and CaCO3 contents might influence the bacterial capacities to solubilize rock phosphate. To fill this objective, we screened the capacity of bacteria isolated from 3 soils to solubilize rock phosphate in vitro in presence of NH4+or NO3-. Then, we selected the most efficient bacterial strains to identify and quantify the release of organic anions into the medium. Among the two hundred and forty-three bacterial strains isolated from the 3 soils, nine and seven isolates were identified with the highest % rock phosphate-solubilization values with NH4+ or NO3- as the sole N-source. Only one strain was able to release free Pi with NH4+ or NO3- as the sole N-source. The most predominant organic acids released by almost all isolates were gluconic acid, lactic acid, glycolic acid, acetic acid, formic acid and pyruvic acid regardless the N-source. However, with NO3- as source of N, the highest concentrations on those acids were found together with the highest release of free Pi into the medium. Molecular analysis of 16S rRNA indicated that almost all strains belonged to Bacillus and Paenibacillus genera. The PCA analysis between soil properties and bacterial capacities to release organic acids and free Pi also revealed that soil factors such as CaCO3 and soil NO3- content positively influenced the release of organic acids by bacteria grown in vitro. Our results concluded that the bacterial rock phosphate-solubilization was intimately related to organic acids production which in turn seemed to be driven by the assimilation of NO3- by bacteria. Therefore, the N-source might be considered a key factor to take into consideration during the screening and selection of suitable strains involved in the P-solubilization.

Potential of Bioassays to Assess Consequences of Cultivation of Acacia mangium Trees on Nitrogen Bioavailability to Eucalyptus Trees: Two Case-Studies in Contrasting Tropical Soils.

We hypothesized that the nitrogen-fixing tree Acacia mangium could improve the growth and nitrogen nutrition of non-fixing tree species such as Eucalyptus. We measured the N-mineralization and respiration rates of soils sampled from plots covered with Acacia, Eucalyptus or native vegetation at two tropical sites (Itatinga in Brazil and Kissoko in the Congo) in the laboratory. We used a bioassay to assess N bioavailability to eucalypt seedlings grown with and without chemical fertilization for at least 6 months. At each site, Eucalyptus seedling growth and N bioavailability followed the same trends as the N-mineralization rates in soil samples. However, despite lower soil N-mineralization rates under Acacia in the Congo than in Brazil, Eucalyptus seedling growth and N bioavailability were much greater in the Congo, indicating that bioassays in pots are more accurate than N-mineralization rates when predicting the growth of eucalypt seedlings. Hence, in the Congo, planting Acacia mangium could be an attractive option to maintain the growth and N bioavailability of the non-fixing species Eucalyptus while decreasing chemical fertilization. Plant bioassays could help determine if the introduction of N2-fixing trees will improve the growth and mineral nutrition of non-fixing tree species in tropical planted forests.

Agroecosystem diversification with legumes or non-legumes improves differently soil fertility according to soil type.

Plant diversification through crop rotation or agroforestry is a promising way to improve sustainability of agroecosystems. Nonetheless, criteria to select the most suitable plant communities for agroecosystems diversification facing contrasting environmental constraints need to be refined. Here, we compared the impacts of 24 different plant communities on soil fertility across six tropical agroecosystems: either on highly weathered Ferralsols, with strong P limitation, or on partially weathered soils derived from volcanic material, with major N limitation. In each agroecosystem, we tested several plant communities for diversification, as compared to a matching low diversity management for their cropping system. Plant residue restitution, N, P and lignin contents were measured for each plant community. In parallel, the soil under each community was analyzed for organic C and N, inorganic N, Olsen P, soil pH and nematode community composition. Soil potential fertility was assessed with plant bioassays under greenhouse controlled climatic conditions. Overall, plant diversification had a positive effect on soil fertility across all sites, with contrasting effects depending on soil type and legumes presence in the community. Communities with legumes improved soil fertility indicators of volcanic soils, which was demonstrated through significantly higher plant biomass production in the bioassays (+18%) and soil inorganic N (+26%) compared to the low diversity management. Contrastingly, communities without legumes were the most beneficial in Ferralsols, with increases in plant biomass production in the bioassays (+39%), soil Olsen P (+46%), soil C (+26%), and pH (+5%). Piecewise structural equation models with Shipley's test revealed that plant diversification impacts on volcanic soil fertility were related to soil N availability, driven by litter N. Meanwhile, Ferralsols fertility was related to soil P availability, driven by litter P. These findings underline the importance of multifactorial and multi-sites experiments to inform trait-based frameworks used in designing optimal plant diversification in agroecological systems.

The grazing activity of Acrobeloides sp. drives phytate mineralisation within its trophic relationship with bacteria.

The microbial loop has been suggested as an alternative route for better utilization of phytate, a poorly available P source to plants. We hypothesized that bacterial grazer activity might dramatically enhance bacterial migration and proliferation, increasing the probability of phytate hydrolysis by bacterial phytases and, thus, phytate mineralization and release of free phosphate. We tested this hypothesis in a two-compartment system with a solid medium containing phytate or free phosphate as the source of P. Two bacterial species, B. subtilis 168 or Bradyrhizobium sp., with or without bacterial grazing nematodes belonging to Acrobeloides sp. previously fed on each of the bacterial species, were inoculated at a single point in the medium. Whatever the P source, nematode migration within both zones allowed the proliferation of bacteria. However, B. subtilis 168 was more efficient in using phytate than Bradyrhizobium sp. since the highest bacterial cell density and free phosphate concentrations were reached by Acrobeloides sp. fed on B. subtilis 168. The grazer activity seemed to be crucial to enhance phytate mineralization, despite Acrobeloides sp. showing a higher preference to feed on Bradyrhizobium sp. This study provides new insights into the effects of bacterial grazer activity on phytate mineralization.

A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach.

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

Introducing N2-fixing trees (Acacia mangium) in eucalypt plantations rapidly modifies the pools of organic P and low molecular weight organic acids in tropical soils.

Many studies have shown that introducing N2-fixing trees (e.g. Acacia mangium) in eucalypt plantations can increase soil N availability as a result of biological N2 fixation and faster N cycling. Some studies have also shown improved eucalypt P nutrition. However, the effects of N2-fixing trees on P cycling in tropical soils remain poorly understood and site-dependent. Our study aimed to assess the effects of planting A. mangium trees in areas managed over several decades with eucalypt plantations on soil organic P (Po) forms and low molecular weight organic acids (LMWOAs). Soil samples were collected from two tropical sites, one in Brazil and one in the Congo. Five different treatments were sampled at each site: monospecific acacia, monospecific eucalypt, below acacias in mixed-species, below eucalypts in mixed-species as well as native vegetation. Po forms and LMWOAs were identified in sodium hydroxide soil extracts using ion chromatography and relationships between these data and available P were determined. At both sites, the concentrations of most Po forms and LMWOAs were different between native ecosystems and monospecific eucalypt and acacia plots. Also, patterns of Po and LMWOAs were clearly separated, with glucose-6-P found mainly under acacia and phytate and oxalate mainly under eucalypt. Despite the strongest changes occurred at site with a higher N2 fixation and root development, acacia introduction was able to change the profile of organic P and LMWOAs in <10 years. The variations between available Pi, Po and LMWOA forms showed that P cycling was dominated by different processes at each site, that are rather physicochemical (via Pi desorption after LMWOAs release) at Itatinga and biological (via organic P mineralization) at Kissoko. Specific patterns of Po and LMWOAs forms found in soil sampled under acacia or eucalypt would therefore explain the effect of acacia introduction in both sites.

How deep can ectomycorrhizas go? A case study on Pisolithus down to 4 meters in a Brazilian eucalypt plantation.

Despite the strong ecological importance of ectomycorrhizal (ECM) fungi, their vertical distribution remains poorly understood. To our knowledge, ECM structures associated with trees have never been reported in depths below 2 meters. In this study, fine roots and ECM root tips were sampled down to 4-m depth during the digging of two independent pits differing by their water availability. A meta-barcoding approach based on Illumina sequencing of internal transcribed spacers (ITS1 and ITS2) was carried out on DNA extracted from root samples (fine roots and ECM root tips separately). ECM fungi dominated the root-associated fungal community, with more than 90% of sequences assigned to the genus Pisolithus. The morphological and barcoding results demonstrated, for the first time, the presence of ECM symbiosis down to 4-m. The molecular diversity of Pisolithus spp. was strongly dependent on depth, with soil pH and soil water content as primary drivers of the Pisolithus spp. structure. Altogether, our results highlight the importance to consider the ECM symbiosis in deep soil layers to improve our understanding of fine roots functioning in tropical soils.

Phosphorus Transport in Mycorrhiza: How Far Are We?

Mycorrhizal fungi considerably improve plant nutrition and help them to cope with changing environments. Particularly, these fungi express proteins to transfer inorganic phosphate (Pi) from the soil to colonized roots through symbiotic interfaces. The mechanisms involved in Pi transfer from fungal to plant cells are still largely unknown. Here, we discuss the recent progress made on the description of these mechanisms and we propose the most promising hypotheses and alternative mechanisms for this process. Specifically, we present a phylogenetic survey of candidate Pi transporters of mycorrhizal fungi that might ensure Pi unload into the symbiotic interfaces. Gathering additional knowledge on mycorrhizal Pi transport will improve the Pi-useefficiency in agroecological systems and will guide towards addressing future research challenges.

HcPT1.2 participates in Pi acquisition in Hebeloma cylindrosporum external hyphae of ectomycorrhizas under high and low phosphate conditions.

Ectomycorrhizal fungi improve tree phosphorus nutrition through transporters specifically localized at soil-hyphae and symbiotic interfaces. In the model symbiosis between the fungus Hebeloma cylindrosporum and the maritime pine (Pinus pinaster), several transporters possibly involved in phosphate fluxes were identified, including three H+:Pi transporters. Among these three, we recently unraveled the function of one of them, named HcPT2, in both pure culture and symbiotic interaction with P. pinaster. Here we investigated the transporter named HcPT1.2, by analyzing inorganic phosphate transport ability in a yeast complementation assay, assessing its expression in the fungus associated or not with the plant, and immunolocalizing the proteins in ectomycorrhizas. We also evaluated the effect of external Pi concentration on expression and localization of HcPT1.2. Our results revealed that HcPT1.2 is involved in Pi acquisition by H. cylindrosporum mycelium, irrespective of the external Pi concentrations.

The Hebeloma cylindrosporum HcPT2 Pi transporter plays a key role in ectomycorrhizal symbiosis.

Through a mutualistic relationship with woody plant roots, ectomycorrhizal fungi provide growth-limiting nutrients, including inorganic phosphate (Pi), to their host. Reciprocal trades occur at the Hartig net, which is the symbiotic interface of ectomycorrhizas where the two partners are symplasmically isolated. Fungal Pi must be exported to the symbiotic interface, but the proteins facilitating this transfer are unknown. In the present study, we combined transcriptomic, microscopy, whole plant physiology, X-ray fluorescence mapping, 32 P labeling and fungal genetic approaches to unravel the role of HcPT2, a fungal Pi transporter, during the Hebeloma cylindrosporum-Pinus pinaster ectomycorrhizal association. We localized HcPT2 in the extra-radical hyphae and the Hartig net and demonstrated its determinant role for both the establishment of ectomycorrhizas and Pi allocation towards P. pinaster. We showed that the host plant induces HcPT2 expression and that the artificial overexpression of HcPT2 is sufficient to significantly enhance Pi export towards the central cylinder. Together, our results reveal that HcPT2 plays an important role in ectomycorrhizal symbiosis, affecting both Pi influx in the mycelium and efflux towards roots under the control of P. pinaster.

Nitrogen and phosphate metabolism in ectomycorrhizas.

1047 I. Introduction 1047 II. Mobilization of soil N/P by ECM fungi 1048 III. N/P uptake 1048 IV. N/P assimilation 1049 V. N/P storage and remobilization 1049 VI. Hyphal N/P efflux at the plant-fungus interface 1052 VII. Conclusion and research needs 1054 Acknowledgements 1055 References 1055 SUMMARY: Nutrient homeostasis is essential for fungal cells and thus tightly adapted to the local demand in a mycelium with hyphal specialization. Based on selected ectomycorrhizal (ECM) fungal models, we outlined current concepts of nitrogen and phosphate nutrition and their limitations, and included knowledge from Baker's yeast when major gaps had to be filled. We covered the entire pathway from nutrient mobilization, import and local storage, distribution within the mycelium and export at the plant-fungus interface. Even when nutrient import and assimilation were broad issues for ECM fungi, we focused mainly on nitrate and organic phosphorus uptake, as other nitrogen/phosphorus (N/P) sources have been covered by recent reviews. Vacuolar N/P storage and mobilization represented another focus point of this review. Vacuoles are integrated into cellular homeostasis and central for an ECM mycelium at two locations: soil-growing hyphae and hyphae of the plant-fungus interface. Vacuoles are also involved in long-distance transport. We further discussed potential mechanisms of bidirectional long-distance nutrient transport (distances from millimetres to metres). A final focus of the review was N/P export at the plant-fungus interface, where we compared potential efflux mechanisms and pathways, and discussed their prerequisites.

In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals.

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

[Suicide in the elderly]. / Le suicide des personnes âgées.

Suicide in the elderly is a real public health issue and has been the subject of a national prevention plan. It is very frequently linked to depression. To prevent people attempting suicide, health professionals must be trained to recognise and manage a suicidal crisis.

A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions.

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid overestimation of 32P efflux into the medium.

Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism.

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the 'Hartig net', where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor 32Phosphate efflux from the fungus previously incubated with 32P-orthophosphate.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Plant Roots Increase Bacterivorous Nematode Dispersion through Nonuniform Glass-bead Media.

Dispersion of bacterivorous nematodes in soil is a crucial ecological process that permits settlement and exploitation of new bacterial-rich patches. Although plant roots, by modifying soil structure, are likely to influence this process, they have so far been neglected. In this study, using an original three-compartment microcosm experimental design and polyvinyl chloride (PVC) bars to mimic plant roots, we tested the ability of roots to improve the dispersion of bacterivorous nematode populations through two wet, nonuniform granular (glass bead) media imitating contrasting soil textures. We showed that artificial roots increased migration time of bacterivorous nematode populations in the small-bead medium, suggesting that plant roots may play an important role in nematode dispersion in fine-textured soils or when soil compaction is high.