Q1. What are the contributions in this paper?
Martins et al. this paper found that mycorrhizal and nonmycizal roots accumulate orthophosphate, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.
A 31P nuclear magnetic resonance study of phosphate levels in roots of ectomycorrhizal and nonmycorrhizal plants of Castanea sativa Mill.
TL;DR: The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhIZal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.
Abstract: 31P-Nuclear Magnetic Resonance (NMR) was used to assess phosphate distribution in ectomycorrhizal and nonmycorrhizal roots of Castanea sativa Mill. as well as in the mycorrhizal fungus Pisolithus tinctorius in order to gain insight into phosphate trafficking in these systems. The fungus P. tinctorius accumulated high levels of polyphosphates during the rapid phase of growth. Mycorrhizal and nonmycorrhizal roots accumulate orthophosphate. Only mycorrhizal roots presented polyphosphates. The content in polyphosphates increased along the 3 months of mycorrhiza formation. In mycorrhizal roots of plants cultured under axenic conditions, the orthophosphate pool decreased along the culture time. In nonmycorrhizal roots the decrease in the orthophosphate content was less pronounced. The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhizal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate.
Summary (2 min read)
Jump to: [Introduction] – [Materials and methods] – [Mycorrhizal synthesis] – [Sample preparation for NMR] – [Phosphate quantification] and [Results and discussion]
Introduction
- The increase in phosphate concentration in mycorrhizal plants was also correlated with increased photosynthetic rates, as the phosphate level of the chloroplast influences photosynthetic rates (Osmond 1981; Coombs 1985; Foyer and Spencer 1986; Jakobsen 1991) .
- It was suggested that phosphate is translocated along hyphae as polyphosphate granules inside vacuoles (Cox et al. 1980 ).
Materials and methods
- Castanea sativa micropropagated plants were obtained as previously described (Martins et al. 1996) .
- The fungus Pisolithus tinctorius (Pers.) Coker and Couch isolate 289/Marx kindly supplied by Dr. Ingrid Kottke (Tübingen University) was maintained on a MMN solid medium (Marx 1969) .
Mycorrhizal synthesis
- Fungus was inoculated 3 weeks before plant transfer.
- In vitro mycorrhization of micropropagated plants was induced 4-5 weeks after plant rooting, by transfer of rooted plants to the petri dishes inoculated 3 weeks before with mycelium.
- A portion of the petri dishes with roots was covered with aluminum foil to prevent oxidation of root phenols by light.
- Control plants were transferred to petri dishes without fungus and maintained under the same conditions.
Sample preparation for NMR
- Samples of excised root systems of mycorrhizal or control plants as well as samples of pure cultures of the fungus were prepared for NMR analysis.
- Similar masses of material and similar packing were used in the different experiments.
- Two glass capillaries were fitted into the NMR tube, one positioned a few millimeters from the bottom (inlet of medium) and the other just above the cotton wool filter that was used to restrict the volume of the biological material (outlet of medium).
- The acquisition time of each spectrum was approximately 40 min.
- In all the experiments, the probe head temperature was kept at 25°C.
Phosphate quantification
- Extraction of total P from roots leaves and stems of mycorrhizal and nonmycorrhizal plants was performed according to Bowman (1988) .
- Phosphate quantification was made 30, 40, 60 and 90 days after mycorrhizal induction in vitro by the molybdenum blue method according to John (1970) .
Results and discussion
- 31 P-NMR spectra of the fungus samples, either in pure culture or extramatrical hyphae collected from petri dishes containing 1-month-old mycorrhizas, showed the presence of resonances due to the vacuolar orthophosphate pool and the polyphosphates pool (Fig. 1 ) as al-ready reported for P. tinctorius (Ashford et al. 1994 ) and for other fungi (Martin et al.
- When present in a P-limiting medium, the stored polyphosphates are hydrolyzed, releasing Pi and maintaining Pi concentration within the fungal cell (Martin et al. 1985; MacFall et al. 1992) .
- Chestnut mycorrhizas are able to accumulate phosphate, mostly in the form of polyphosphates, in contrast to nonmycorrhizal roots, which accumulate only orthophosphate (Fig. 2 ).
- The differences in phosphate content between mycorrhizal and nonmycorrhizal plants decreased with time.
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Figures (2)
Fig. 2 31P-NMR spectra of control (A) and mycorrhizal (B) chestnut roots at different mycorrhization times: (a) 3 weeks after mycorrhizal induction; (b) 1 month after mycorrhizal induction; (c) 3 months after mycorrhizal induction. Pi, intracellular orthophosphate; polyP, polyphosphates&/fig.c: 
Fig. 1 31P-NMR spectra of the fungus Pisolithus tinctoriusgrown in axenic conditions with different ages: (a) 7 days, (b) 15 days and (c) 1.5 months. Pi, intracellular orthophosphate; tP, terminal phosphate; UDP-Hexose, uridine diphosphate hexose; PP, penultimate phosphate; polyP, polyphosphates&/fig.c:
Citations
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TL;DR: While large gaps remain in understanding of the physiological and molecular mechanisms that underpin movement of P in ECM mycelia in soil and P transfer to the plant, host P demand seems likely to be a key driver of these processes.
Abstract: Many forest trees have evolved mutualistic symbioses with ectomycorrhizal (ECM) fungi that contribute to their phosphorus (P) nutrition. Forest productivity is frequently limited by P, a phenomenon that is likely to become more widespread under future conditions of elevated atmospheric CO2 concentration [CO2]. It is thus timely that this review considers current understanding of the key processes (absorption, translocation and transfer to the plant host) in ECM fungus-mediated P nutrition of forest trees. Solubilisation of inorganic P (Pi) and hydrolysis of organic P by ECM fungi in soil occurs largely at the growing mycelial front, where Pi absorption is facilitated by high affinity transporters. While large gaps remain in our understanding of the physiological and molecular mechanisms that underpin movement of P in ECM mycelia in soil and P transfer to the plant, host P demand seems likely to be a key driver of these processes. ECM fungi may make considerable contributions to meeting the likely increased P demand of trees under elevated [CO2] via increased colonization levels, shifts in ECM fungal community structure and changed patterns of EMM production. Further research into the spatial scale of ECM-mediated P movements in soil, along with the interplay between ECM fungi and other soil microflora is advocated.
129 citations
Cites background from "A 31P nuclear magnetic resonance st..."
...…with factors that include P availability and the relative growth rates of the host and ECM fungal partner, but as much as 90% of absorbed P can be retained in the fungal tissues of excised ECM root tips, mainly in the form of polyphosphates (Harley and McCready 1952, 1981; Martins et al. 1999)....
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TL;DR: NMR has been useful in analysing metabolism, transport and energetics, and the results of such studies have practical and ecological significance.
Abstract: Summary
Nuclear magnetic resonance (NMR) studies of mycorrhizal symbioses have illuminated a number of functional aspects of these complex associations. Here we review studies of the two main types of mycorrhiza (ectomycorrhizas and arbuscular mycorrhizas) to which NMR has been applied. Although the physiological questions addressed in each case are frequently the same, these two mutualistic symbioses are sufficiently different to justify separate discussion. In conjunction with isotopic labelling NMR is able to examine the transfer of substrates between the symbionts both in vivo and in vitro, as well as the production of secondary metabolites in response to colonization. In addition, this methodology is capable of determining the locations of the biosynthesis and translocations of storage compounds, such as polyphosphates, lipids and carbohydrates, in mycorrhizal fungi both in the free-living and in the symbiotic stages of their life cycle. NMR has been useful in analysing metabolism, transport and energetics, and the results of such studies have practical and ecological significance. Models of transport and physiology to which NMR has contributed form the necessary foundation for functional genomic exploration.
63 citations
TL;DR: The hypothesis that polyP is the major P species translocated in the tubular vacuolar network, the presence of which was previously demonstrated in AM fungi, is supported.
Abstract: Summary
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Polyphosphate (polyP) is presumably central to phosphate (P) metabolism of arbuscular mycorrhizal (AM) fungi, but its synthesis, location and chain lengths are poorly characterized. Here, we applied noninvasive and nondestructive nuclear magnetic resonance (NMR) spectroscopy to obtain novel information on AM fungal polyP.
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In vivo31P NMR spectroscopy was used to characterize polyP and other P pools in external hyphae and in mycorrhizal roots of associations between Glomus intraradices and cucumber (Cucumis sativus).
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A time-course study of P-starved external hyphae supplied with additional P showed that polyP appeared more rapidly than vacuolar inorganic P. These P metabolites also appeared in the roots, but later. PolyP considerably exceeded amounts of vacuolar inorganic P, where it was located in acidic, presumably vacuolar compartments, and had a short average chain length.
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The rapid synthesis of polyP might be important for the maintenance of effective hyphal P uptake. Our data support the hypothesis that polyP is the major P species translocated in the tubular vacuolar network, the presence of which was previously demonstrated in AM fungi.
62 citations
TL;DR: The 31P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in theSpectra of nonmycorrhIZal roots, demonstrating that the Pi taken up by the fungus was transformed into PolyP with a short chain length.
Abstract: 31P nuclear magnetic resonance (NMR) spectroscopy was used to study phosphate (P) metabolism in mycorrhizal and nonmycorrhizal roots of cucumber (Cucumis sativus L) and in external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. The in vivo NMR method allows biological systems to be studied non-invasively and non-destructively. 31P NMR experiments provide information about cytoplasmic and vacuolar pH, based on the pH-dependent chemical shifts of the signals arising from the inorganic P (Pi) located in the two compartments. Similarly, the resonances arising from α, β and γ phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with this method. The 31P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in the spectra of nonmycorrhizal roots. This demonstrated that the Pi taken up by the fungus was transformed into PolyP with a short chain length. The spectra of excised AM fungi revealed only a small signal from the cytoplasmic Pi, suggesting a low cytoplasmic volume in this AM fungus.
56 citations
Cites background from "A 31P nuclear magnetic resonance st..."
...…role in the transportation and storage of phosphorus in the fungus, and when P was added to ectomycorrhizal fungi, PolyP signals were found in the31P NMR spectra (Ashford et al., 1994; Gerlitz and Gerlitz, 1997; Gerlitz and Werk, 1994; Martin et al., 1983, 1985, 1994; Martins et al., 1999)....
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TL;DR: The results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots, and specifically increased the proportion of short-chain polyP in the interacting mycelia.
Abstract: 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 32P labelling to quantify P accumulation and P efflux and in vivo and in vitro 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 32P release compared to 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.
21 citations
Cites methods from "A 31P nuclear magnetic resonance st..."
...Signal identification was carried out according to chemical shifts reported in other ectomycorrhizal fungi (Gerlitz & Werk 1994; Martins et al. 1999)....
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References
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TL;DR: It is proposed that polyphosphate is transported along the hyphae by the motile tubule and vacuole system present in this fungus.
Abstract: SUMMARY Polyphosphate was extracted from an Australian isolate of Pisolithus tinctorius (Pers.) Coker & Couch, known to be mycorrhizal with Eucalyptus pilularis. It was separated from RNA and characterized by PAGE and 31P NMR spectroscopy. A broad pink band, which showed y-metachromasy with toluidine blue, ran faster on acrylamide gels than a number of purple-blue bands (various RNA fractions) and did not stain with ethidium bromide, was identified as polyphosphate. The major pink band co-migrated with a synthetic polyphosphate standard of a chain length of about 15 phosphate (Pi) subunits, and analysis by 31P nuclear magnetic resonance (NMR) spectroscopy confirmed that polyphosphate was a major fraction in the extract. Polyphosphate was extracted from mycelium grown at both high and low levels of Pi, but the amount extracted from cultures grown with 10 mm Pi (added as ammonium phosphate) was significantly greater. Polyphosphate was a substantial fraction of the phosphorus present in the hyphae and it is proposed that polyphosphate is transported along the hyphae by the motile tubule and vacuole system present in this fungus.
51 citations
TL;DR: The results are discussed in relation to phosphate accumulation and translocation by the extramatrical mycelium of ectomycorrhizas in soil.
50 citations
TL;DR: Four ectomycorrhizal fungi (Amanita muscaria, Laccaria laccata, Piloderma croceum and Pisolithus tinctorius) were used to produce mycorrhiza on seedlings and micropropagated plants of Castanea sativa in vitro.
Abstract: Four ectomycorrhizal fungi (Amanita mus- caria, Laccaria laccata, Piloderma croceum and Pisoli- thus tinctorius) were used to produce mycorrhiza on seedlings and micropropagated plants of Castanea sati- va in vitro. Pisolithus tinctorius was most effective in colonizing roots of both micropropagated plants and seedlings. A. muscaria and L. laccata only colonized a few feeder roots of some plants and Piloderma croceum did not form mycorrhizas. Mycorrhization of micropro- pagated plants increased survival and growth during weaning.
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TL;DR: Mycorrhizal micropropagated Castanea sativa plants were studied in terms of growth and physiological parameters following in vitro mycorrhization with Pisolithus tinctorius to enhance the acclimatization process.
Abstract: Mycorrhizal micropropagated Castanea sativa plants were studied in terms of growth and physiological parameters following in vitro mycorrhization with Pisolithus tinctorius. Mycorrhization enhanced growth of micropropagated chestnut plants, increased their protein content and photosynthetic rates, decreased the respiratory rates and CO2 compensation point. RuBisCO activity was not significantly different in mycorrhizal and control plants, although there was an increase in the amount of RuBisCO in the former. Mycorrhization increased plant biomass and improved plants physiological status, thus enhancing the acclimatization process.
45 citations
TL;DR: This chapter provides a framework for some practical approaches to the study of carbon metabolism in mycorrhiza and shows that some problems may be investigated by means of methods already developed.
Abstract: Publisher Summary This chapter provides a framework for some practical approaches to the study of carbon metabolism in mycorrhiza. The ability of a fungus to utilize different carbon compounds may be tested by growing the fungus on agar or in liquid medium. It is shown that some problems may be investigated by means of methods already developed. Among these are enzymatic studies of hexose catabolism in the arbuscular fungi including the possibly important role of dark C0 2 fixation providing anaplerotic pathways to Krebs cycle intermediates, as well as 14 Cstudies of below-ground carbon allocation, carbon incorporation by the external hyphae and interplant carbon flow mediated by connecting hyphal networks. There is an obvious need for comparative work using well-defined species of both plants and fungi. Other important areas are methodologically less well developed. These include studies of mechanisms involved in the host-fungus carbon transfer and direct measurements of host -fungus carbon flow. Approaches already developed for the study of other symbiotic associations may be an important source of new experimental initiatives in mycorrhizal research.
42 citations