Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo31P NMR spectroscopy

Authors


Author for correspondence: Iver Jakobsen Tel: +45 46 774154 Fax: +45 46 774202 Email: iver.jakobsen@risoe.dk

Summary

  • • 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.
  • • 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).
  • • 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.
  • • 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.

Introduction

Phosphate (P) is an essential nutrient for all organisms, is required in relatively large amounts and is often limiting to plant growth. Plants therefore have mechanisms for uptake of this nutrient with high effectiveness. Arbuscular mycorrhizal (AM) fungi colonize the roots of most land plants and the symbiosis between them is characterized by bidirectional nutrient transport (Smith & Read, 1997). The AM fungus receives an indispensable supply of fixed carbon from the host and mediates a soil-to-plant transport of mineral nutrients. The external mycelium of AM fungi allows the plant to access inorganic orthophosphate (Pi) in the soil solution beyond the depletion zone formed around the actively absorbing roots.

The uptake, translocation and transfer of P by the external mycelium of the AM fungi have been studied extensively, and a model of the overall mechanisms has been widely accepted. It is believed that Pi in the soil solution is absorbed by the external mycelium via an AM fungal P transporter energized by a P-type H+-ATPase (Harrison & van Buuren, 1995; Ferrol et al., 2000; Maldonado-Mendoza et al., 2001). The Pi entering the cytoplasm of the AM fungus may be incorporated into phosphorylated primary metabolites, structural molecules and nucleic acids. It is assumed that excess Pi taken up into the external hyphae is subsequently transferred to the vacuoles and condensed into polyphosphate (polyP). The P-containing substances, including polyP, are then translocated to the intraradical hyphae in vacuoles in a motile tubular vacuolar system similar to that of ectomycorrhizas (Smith & Read, 1997). Recent studies of AM fungi have confirmed the presence of tubular vacuoles and microtubules (Timonen et al., 2001; Uetake et al., 2002). Once translocated to the symbiotic interface inside the root, the polyP has to be hydrolysed and the Pi released and transferred to the plant root cells. This transfer is believed to occur at the arbuscular interface, which is in agreement with the recent discovery that plant P transporters are expressed in root cells containing arbuscules (Rosewarne et al., 1999; Rausch et al., 2001; Harrison et al., 2002). The efflux of Pi and concurrent decrease of polyP in the hyphae of Gigaspora margarita appears to be promoted by increased glucose supply, indicating a role for polyP in the exchange of carbon and Pi between symbionts (Solaiman & Saito, 2001).

PolyP has a clear osmotic advantage over Pi and synthesis of polyP may also be a major part of the mechanism by which the fungus controls the cytoplasmic Pi concentration (Mimura, 1999). PolyP has been detected in AM fungi by histochemical methods (Cox et al., 1975; Cox et al., 1980; Boddington & Dodd, 1999; Ezawa et al., 2001), by extraction methods followed by polyacrylamide gel electrophoresis (PAGE) (Callow et al., 1978; Solaiman et al., 1999), by a polyP kinase assay (Ezawa et al., 2004) and by nuclear magnetic resonance (NMR) (Shachar-Hill et al., 1995; Rasmussen et al., 2000). However, details of the metabolism of polyP and its role in symbiotic P supply are still unclear, in particular the amount, size and major role of polyP present in the external and intraradical hyphae. Several investigations suggest the presence of very long-chain polyP or even granules located in the external mycelium (Callow et al., 1978; Solaiman et al., 1999), supporting the idea that polyP metabolism in external and intraradical hyphae may be different. There may also be differences between fungal species, as the external mycelium of Glomus manihotis seemed not to accumulate polyP, whereas high amounts were observed in Gigaspora rosea (Boddington & Dodd, 1999).

Overall, the presence of polyP in many species of AM fungi is well documented, but the characterization of the polyP and the mechanisms involved in its metabolism are unclear. Staining methods with variable specificity or invasive methods have commonly been used to identify polyP and artefacts of specimen preparation could possibly have interfered with accurate determination of the polyP chain length (Orlovich & Ashford, 1993). Noninvasive and nondestructive techniques are required to obtain more detailed information of P pools and polyP content in AM fungi. For this purpose, in vivo31P NMR spectroscopy is a unique analytical method (Rasmussen et al., 2000; Ratcliffe & Shachar-Hill, 2001).

The objectives of the present study were to characterize the dynamics of incorporation of Pi into various P pools within external mycelium and mycorrhizal roots and in particular to investigate the dynamics of polyP synthesis, its cellular location and chain lengths. A time-course study was carried out using in vivo31P NMR to investigate the formation of P compounds in differently P-treated hyphae of the AM fungus Glomus intraradices and mycorrhizal cucumber (Cucumis sativus) roots. Chain lengths of polyP were further investigated by the use of extraction procedures followed by colorimetric measurements and 31P NMR.

Materials and Methods

Biological materials, soil and overall experimental design

The AM fungus G. intraradices Schenck & Smith (DAOM 197198, Biosystematics Research Centre, Ottawa, Canada) was used in all experiments and was grown in symbiosis with C. sativus L. (cv. Aminex, F1 hybrid). External mycorrhizal hyphae were produced in a compartmented growth system, where the hyphae could be rapidly extracted from quartz sand. The growth system consisted of a 75 mm diameter 25-µm nylon mesh bag filled with 725 g of an irradiated (10 kGy, 10 MeV electron beam) 1 : 1 moraine soil and quartz sand mixture (w : w, here called ‘soil’) into which was incorporated 75 g of G. intraradices inoculum from a Trifolium subterraneum L. pot culture. Basal nutrients minus P were mixed into the soil in the following amounts (mg kg−1 dry soil): K2SO4, 75.0; CaCl2·5H2O, 75.0; CuSO4·5H2O, 2.1; ZnSO4·7H2O, 5.4; MnSO4·H2O, 10.5; CoSO4·7H2O, 0.39; NaMoO4·2H2O, 0.18; MgSO4·7H2O, 45.0. This final soil had a pH(H2O) of 6.7 and a 0.5 m NaHCO3-extractable P content of 11 µg P g−1 (Olsen et al., 1954). The nylon mesh bag was placed in the centre of a pot filled with 2200 g of washed, autoclaved quartz sand (Johansen et al., 1996). Inoculum was incubated for 1 wk in moist (60% of water holding capacity) soil and three pregerminated seeds were sown in the nylon mesh bag. After seedling emergence, plants were thinned to two per pot and the pots were placed in a growth chamber with a 16 h light/8 h dark cycle at 20°C/16°C and Osram daylight lamps (HQI T250 W/D 500 µmol m−2 s−1). The pots were watered daily and an aqueous solution of 0.36 m NH4NO3 was supplied to the pots weekly to provide a total addition of 200 mg N per pot. The sand in the hyphal compartment was replaced with fresh washed and autoclaved sand four weeks after sowing and the hyphae were harvested from this sand after another 2 wk plus the time after additional P treatment. The experiment included 24 pots in total.

Phosphorus treatments and harvest

The experiment included three P treatments, as outlined in Table 1. At 0 h, each of 20 pots received 100 mg P as an aqueous solution of KH2PO4 along the outer edge of the hyphal compartment, which had previously received either no P (treatment 1, 10 pots) or a 0.7 mm P nutrient solution (pH 6.0, 1 mm Ca(NO3)2·4H2O, 1 mm NH4NO3, 1 mm K2SO4, 0.8 mm MgSO4·7H2O, 0.7 mm Na2HPO4·2H2O, 25 µm Fe(III)Na-ethylenediaminetetraacetic acid (EDTA), 25 µm H3BO3, 5 µm MnSO4·H2O, 2 µm ZnSO4·7H2O, 0.5 µm CuSO4·5H2O, 0.1 µm Na2MoO4·2H2O, 4 nm CoCl2·6H2O) during the previous 2 wk (treatment 2, 10 pots). Treatment 2 served as a reference for the study of P dynamics in treatment 1. Three additional pots received no P at 0 h, and received only the 0.7 mm P nutrient solution during the previous 2 wk (treatment 3, three pots). One pot received no P at all and served as control. Pots were harvested sequentially (see Table 1) and this time-course approach was preferred over the harvest of replicate pots from individual treatments in order to focus on the P pool dynamics in response to the various P treatments.

Table 1.  Experimental P treatments and fresh weights of external mycelium of Glomus intraradices and mycorrhizal cucumber (Cucumis sativus) plants
 Harvest (hours after P supply at 0 h)Average value
12510162434487296
  1. Control pot (No P added, harvest at time 96 h): shoot f. wt, 26.3 g; root f. wt, 20.1 g; hyphae f. wt, 0.10 g. NMR, nuclear magnetic resonance.

Treatment 1: 100 mg P at 0 h; no previous P supply
Total P supply (mg)100100100100100100100100100100 
Shoot f. wt (g) 30.0 18.6 23.8 25.2 21.3 25.7 25.5 26.1 29.4 33.326
Root f. wt (g) 19.2 11.4 13.6 11.4 12.0 28.5 17.6 17.7 18.5 14.016
Root f. wt in NMR tube (g)  0.14  0.09  0.13  0.12  0.16  0.17  0.26  0.17  0.25  0.17 
Hyphae f. wt (g)  0.08  0.03  0.12  0.03  0.12  0.26  0.73  0.13  0.70  0.07 0.23
Hyphae f. wt in NMR tube (g)  0.08  0.03  0.12  0.03  0.12  0.11  0.12  0.07  0.14  0.07 
Treatment 2: 100 mg P at 0 h; nutrient solution with 0.7 mm P during previous 2 wk
Total P supply (mg)125122124124120119120127132130 
Shoot f. wt (g) 40.9 36.0 41.2 39.9 33.8 30.5 39.0 42.0 53.2 41.340
Root f. wt (g) 32.0 22.6 27.5 30.0 23.2 20.7 18.1 27.3 33.4 22.426
Root f. wt in NMR tube (g)  0.31  0.12  0.16  0.20  0.10  0.21  0.32  0.25  0.40  0.11 
Hyphae f. wt (g)  0.52  0.18  0.39  0.44  0.46  0.26  0.35  0.38  0.51  0.12 0.36
Hyphae f. wt in NMR tube (g)  0.07  0.08  0.09  0.08  0.06  0.08  0.07  0.07  0.08  0.06 
Treatment 3: No P at 0 h; nutrient solution with 0.7 mm P during previous 2 wk
Total P supply (mg) 20    24     32 
Shoot f. wt (g) 39.4    33.3     49.641
Root f. wt (g) 28.9    17.9     24.324
Root f. wt in NMR tube (g)  0.16     0.14      0.15 
Hyphae f. wt (g)  0.38     0.03      0.08 0.16
Hyphae f. wt in NMR tube (g)  0.09     0.03      0.08 

External hyphae of individual pots were collected on a 38-µm sieve in three cycles of aqueous suspension of the sand and subsequent decanting. The hyphae samples were gently rinsed, first in water and then in a buffer containing 10% (v : v) D2O, 50 mm glucose, 10 mm 2-(N-morpholino) ethanesulfonic acid (MES) and 0.1 mm CaSO4 at pH 6.0 to remove most of the remaining sand. This harvest and rinsing procedure lasted about 15 min per pot. Root material was carefully collected from the mesh bag after washing away the soil and the excised root pieces were placed in a buffer similar to the one used for the hyphae. Subsamples of hyphae or roots were packed loosely in an NMR tube, using similar masses of material (between 0.03 and 0.14 g f. wt hyphae and 0.09–0.40 g f. wt roots, respectively) and similar packing density in the different experiments.

Plant analysis

The total shoot fresh weights were determined. The total fresh weights of both hyphae and roots were determined after NMR analysis after removing excess moisture using filter paper. Fresh root subsamples were cleared in 10% (w : v) KOH and stained with 0.05% (w : v) Trypan blue by a modification of the method of Kormanik & McGraw (1982) and the percentage of root lengths colonized by G. intraradices were measured in accordance with Giovannetti & Mosse (1980).

In vivo NMR experiments

The in vivo31P spectra were recorded at 242.812 MHz on a Varian Unity Inova 600 spectrometer (Varian Instruments, Palo Alto, CA, USA) with a superconducting magnet (Oxford Instruments, Oxford, UK) using a broadband 10-mm diameter probe head. The spectra were accumulated with a 45° pulse angle (26.5 µs), an acquisition time of 0.064 s, a recycle time of 0.45 s, proton decoupling by Waltz-16 composite pulse sequence, a sweep width of 15.0 kHz, 12 000 scans, a total acquisition time of 105 min and processed with 30 Hz line broadening. All in vivo spectra were recorded using an airlift system operating with an oxygen flow rate of c. 90 ml min−1 (Fox et al., 1989), with the hyphae or roots placed in the same buffer as used during the washing procedure. The chemical shifts of the signals in the 31P NMR spectra were measured relative to the signal from 100 mm methylene diphosphonic acid (MDP, pH 7.5) contained in a capillary included in the NMR tube, and the chemical shifts were quoted on the scale that puts the signal from 85% (w : v) orthophosphoric acid at 0 ppm. Assignment of the various P signals in the spectra was done by comparison with 31P NMR spectra of ectomycorrhizal fungi (Martin et al., 1983, 1994; Grellier et al., 1989; Gerlitz, 1996), roots colonized by ectomycorrhizal fungi (Loughman & Ratcliffe, 1984; MacFall et al., 1992; Gerlitz & Werk, 1994; Martins et al., 1999), AM fungi and roots colonized by AM fungi (Shachar-Hill et al., 1995; Rasmussen et al., 2000). Estimates of the cytoplasmic and vacuolar pH were obtained from the chemical shift of the cytoplasmic or vacuolar Pi signal using calibration curves obtained from solutions with ionic compositions typical of the cytosol and the vacuole (Spickett et al., 1993). Similarly, a calibration curve for the chemical shift of terminal polyP vs pH was made in order to determine the pH of the compartment in which the polyP was located (Fig. 1). The calibration solution contained 5 mm polyP glass of type 25 (Sigma, St Louis, MO, USA), 2 mm MgCl2 and 100 mm KCl (Martin et al., 1994).

Figure 1.

Titration curve for the chemical shift of terminal polyphosphate (polyP) as a function of pH. The polyP glass was present at 5 mm and had an estimated chain length of 22 residues.

Extraction procedures, colorimetric measurements and NMR of extracts and standards

The 31P NMR spectra of synthetic polyP glasses of types 5, 15, 25, 35 and 75+ (obtained from Sigma) were recorded in order to investigate the upper limit of NMR-visible polyP (i.e. the maximum average chain length that can be observed in an NMR spectrum). Approximately 5 mg of the synthetic polyP was dissolved in 2.7 ml H2O and 0.3 ml D2O with 0.1 m Na2EDTA added to obtain sharper signals in the NMR spectra (MacDonald & Mazurek, 1987). 31P NMR spectroscopy was performed using the same spectrometer as used for in vivo measurements. The spectra were accumulated with a 90° pulse angle (53 µs), an acquisition time of 0.59 s, a recycle time of 2.5 s, proton decoupling by Waltz-16 composite pulse sequence, a sweep width of 15.0 kHz, 1200 scans, a total acquisition time of 61 min and processed with 20 Hz line broadening. The chemical shifts of the signals in the 31P NMR spectra were measured relative to the signal from 100 mm MDP (pH 7.5) contained in a capillary included in the NMR tube, and the chemical shifts were quoted on the scale that places the signal from 85% (w : v) orthophosphoric acid at 0 ppm.

Different polyP fractions in the hyphae used for in vivo31P NMR investigations were successively extracted using the method of Clark et al. (1986). Samples were first homogenized in a mortar in ice-cold trichloroacetic acid (TCA), which extracted the acid-soluble, short-chain polyP. Subsequent extraction with EDTA and phenol–chloroform (PC) extracted the neutral-soluble long-chain polyP and the long-chain granular polyP, respectively. Extracts were made from four combined samples of hyphae: (1) treatment 1, harvests 1–5 h; (2) treatment 1, harvests 10–96 h; (3) treatment 2, harvests 1–5 h; and (4) treatment 2, harvests 10–96 h. This combination was introduced to reduce number of extractions and the selected time frames ensured that treatment 1 samples with extremely low NMR signals were kept separate from the others. The extracted polyP in aqueous solution was precipitated by adding Tris-HCl (1 m, pH 7.6) to a final concentration of 0.2 m and two volumes of acetone. The mixture was frozen at −80°C for more than 15 min, thawed and centrifuged for 10 min. The residue was air-dried over night, dissolved in water and kept at −20°C until analysis.

The polyP content in the extracts was identified by measuring the metachromatic reaction of toluidine blue at 530 nm and 630 nm, according to Griffin et al. (1965) and Solaiman et al. (1999). The assay was performed by adding 10 µl of the polyP extract to tubes containing 0.75 ml each of 0.2 n acetic acid and 30 mg l−1 toluidine blue. The content of polyP was estimated by comparison of the absorption spectra with standard curves (see Fig. 8a) produced by using 1 µg and 5 µg of each of three synthetic polyP glasses; the polyP chosen were type 5 and type 25 polyP for the short-chain and type 75+ polyP for the long-chain polyP. PolyP was classified as being present or not in the different fractions.

Figure 8.

In vivo31P nuclear magnetic resonance (NMR) spectra of mycorrhizal cucumber (Cucumis sativus) roots from sequentially harvested pots, which had received no P at 0 h, but a nutrient solution with 0.7 mm P during the previous 2 wk (treatment 3). Fresh weight of samples in the NMR tube varied between 0.14 g and 0.16 g and did not include all harvested material. Spectra were calibrated against a reference signal. Peak assignments were as follows: a, several phosphomonoesters; b, cytoplasmic inorganic orthophosphate (Pi); c, vacuolar Pi; d, γ-NTP; e, β-NDP/terminal polyphosphate (polyP) residues; f, α-NTP; g, uridine diphosphoglucose; h, β-NTP; i, penultimate polyP residues; j, central polyP residues.

The 31P NMR spectroscopy of the polyP-containing extracts was performed using the same parameters as used with synthetic polyP, except that the recycle time was 6 s, given a total acquisition time of 132 min and the spectra were processed with 10 Hz line broadening. Each extract was diluted to 3.1 ml with water containing 10% (v : v) D2O.

The effectiveness of TCA extraction of short-chain polyP was further studied by extracting synthetic polyP type at two concentrations (10.4 mg and 1.1 mg) and by investigation of the extracts by 31P NMR. Each extract was diluted to 3.1 ml with water containing 10% (v : v) D2O and the spectra were obtained using the same parameters as used for extracts of hyphae. The extracted P was hydrolysed in a solution of nitric–perchloric acid (4 : 1, v : v) and total P content was determined by the molybdate blue method (Murphy & Riley, 1962).

Results

Plant growth and mycorrhiza formation

Biomass data for the cucumber plants and G. intraradices are presented in Table 1. Cucumber plants from pots that had received no previous P supply (Table 1, treatment 1) were smaller than plants that had received P during the previous 2 wk (Table 1, treatments 2 and 3). This difference was clear for shoot and root f. wts, and the mass of the external hyphae showed the same trend. The biomass components showed no clear response to the addition of P at 0 h (Table 1, treatments 2 and 3), but their magnitude varied and only low quantities of mycelium could be harvested from some pots (Table 1). Although statistics were not applicable because individual pots received different amounts of P, average biomass values for each of the three sequential harvests are included in Table 1. The proportion of root length colonized by mycorrhizal fungi was in the range 69–97% (mean = 85%) and was independent of P treatment.

Phosphate pools in external hyphae

In vivo31P NMR spectra for external hyphae harvested from each pot are presented in Figs 2–4 and spectra are marked ‘High amount of hyphae’ or ‘Low amount of hyphae’ when hyphal f. wts differed markedly from the average. The signal at −23.0 ppm corresponded to the central Pi residues in a NMR-visible polyP chain. This signal was present in all spectra obtained from hyphae from treatments 2 and 3 (Figs 3 and 4, peak d), but did not appear in hyphae from treatment 1 until between 2 h and 5 h after the P supply at 0 h (Fig. 2, peak d). The three remaining signals visible in the spectra were assigned to vacuolar Pi (peak a, 0.4 ppm), terminal Pi residues in the polyP chain (peak b, −6.4 ppm) and penultimate Pi residues in the polyP chain (peak c, −20.1 ppm). The vacuolar Pi signal was not detectable until 10 h after P was supplied and the terminal and penultimate Pi residue signals of the polyP chain could not be detected until 16 h after P supply within treatment 1 (Fig. 2). No signals were visible in the spectrum of hyphae from the control pot (no P added, spectrum not shown). The chemical shift value of the vacuolar Pi signal based on calibration curves (see the Materials and Methods section) corresponded to a vacuolar pH of approximately 5.5, independent of harvest time or P treatment. The chemical shifts of terminal Pi residues in the polyP chain predict a pH of approximately 6.0, as estimated from the pH titration curve for terminal Pi residues (Fig. 1). This acidic pH value indicated that the NMR-visible polyP was present in a vacuolar compartment.

Figure 2.

In vivo31P nuclear magnetic resonance (NMR) spectra of excised Glomus intraradices hyphae from sequentially harvested pots, which had received 100 mg P per pot at 0 h, but no previous P supply (treatment 1). Fresh weight of samples in the NMR tube varied between 0.03 g and 0.14 g and did not include all harvested material. Spectra were calibrated against a reference signal. Peak assignments were as follows: a, vacuolar inorganic orthophosphate (Pi); b, terminal polyphosphate (polyP) residues; c, penultimate polyP residues; d, central polyP residues.

Figure 3.

In vivo31P nuclear magnetic resonance (NMR) spectra of excised Glomus intraradices hyphae from sequentially harvested pots, which had received 100 mg P per pot at 0 h and a nutrient solution with 0.7 mm P during the previous 2 wk (treatment 2). Fresh weight of samples in the NMR tube varied between 0.06 g and 0.09 g and did not include all harvested material. Spectra were calibrated against a reference signal. Peak assignments were as follows: a, vacuolar inorganic orthophosphate (Pi); b, terminal polyphosphate (polyP) residues; c, penultimate polyP residues; d, central polyP residues.

Figure 4.

In vivo31P nuclear magnetic resonance (NMR) spectra of excised Glomus intraradices hyphae from sequentially harvested pots, which had received no P at 0 h, but a nutrient solution with 0.7 mm P during the previous 2 wk (treatment 3). Fresh weight of samples in the NMR tube varied between 0.03 g and 0.09 g and did not include all harvested material. Spectra were calibrated against a reference signal. Peak assignments were as follows: a, vacuolar inorganic orthophosphate (Pi); b, terminal polyphosphate (polyP) residues; c, penultimate polyP residues; d, central polyP residues.

Vacuolar Pi and polyP were quantified from the areas under the peaks for vacuolar Pi and central polyP residues. There was approximately 10 times more polyP within the same hyphal sample as vacuolar Pi, independent of P treatment (Figs 2–4). Also, vacuolar Pi tended to increase to a constant level in treatments 1, 2 and 3. The amounts of polyP tended to increase with time after additional P supply, not only in treatment 1 with no previous P supply, but also in treatment 2, although there was considerable variation in the areas of the peaks for central polyP residues (Figs 2 and 3). The areas of the peaks also varied for central polyP residues in the spectra of hyphae from treatment 3 (Fig. 4) and this variation corresponded to the variation in quantity of hyphae used (see also Table 1).

Phosphate pools in mycorrhizal cucumber roots

A time-course of in vivo31P NMR spectra was also obtained for the mycorrhizal roots from the three P treatments. The spectrum of roots from the 24-h harvest of treatment 2 was enlarged for assignment of the various P signals (Fig. 5). This spectrum contained major signals at −22.9 ppm and 0.3 ppm from polyP (peak j) and vacuolar Pi (peak c), respectively, as well as signals from several other P-containing analytes. The signals around 4.4 ppm corresponded to several phosphomonoesters (peak a) and the smaller signal at 2.3 ppm was attributed to cytoplasmic Pi (peak b). Various signals for nucleic acid triphosphates (NTP, peaks d, f and h) at −5.3 ppm, −10.4 ppm and −19.0 ppm were also easily distinguished, together with signals from uridine diphosphoglucose (peak g) at around −11.0 ppm and −12.5 ppm. Signals from terminal (peak e) and penultimate (peak i) Pi residues of polyP could also be detected at −6.4 ppm and −20.1 ppm. However, these signals overlapped the signals from phosphates of NTP.

Figure 5.

In vivo31P nuclear magnetic resonance (NMR) spectrum of mycorrhizal cucumber (Cucumis sativus) roots harvested 24 h after the supply of 100 mg P; the pot had received a nutrient solution with 0.7 mm P during the previous 2 wk (treatment 2). The spectrum was magnified in order to show details and peak assignments were as follows: a, several phosphomonoesters; b, cytoplasmic inorganic orthophosphate (Pi); c, vacuolar Pi; d, γ-NTP; e, β-NDP/terminal polyphosphate (polyP) residues; f, α-NTP; g, uridine diphosphoglucose; h, β-NTP; i, penultimate polyP residues; j, central polyP residues.

No P signals could be detected in the spectra of mycorrhizal roots from treatment 1 until 10 h after P supply, when vacuolar Pi and the central Pi residues of polyP could be identified (Fig. 6). Later spectra of treatment 1 roots contained all the P signals, which could be detected already at the 1-h harvest in roots previously receiving P (treatments 2 and 3). No signals could be detected from roots of the control pot, which had received no P at all (spectrum not shown).

Figure 6.

In vivo31P nuclear magnetic resonance (NMR) spectra of mycorrhizal cucumber (Cucumis sativus) roots from sequentially harvested pots, which had received 100 mg P per pot at 0 h, but no previous P supply (treatment 1). Fresh weight of samples in the NMR tube varied between 0.09 g and 0.26 g and did not include all harvested material. Spectra were calibrated against a reference signal. Peak assignments were as follows: a, several phosphomonoesters; b, cytoplasmic Pi; c, vacuolar inorganic orthophosphate (Pi)i; d, γ-NTP; e, β-NDP/terminal polyphosphate (polyP) residues; f, α-NTP; g, uridine diphosphoglucose; h, β-NTP; i, penultimate polyP residues; j, central polyP residues.

The positions of the signals for vacuolar and cytoplasmic Pi did not change between harvest times or P treatments; pH was estimated to be 7.4 and approximately 5.3 in the cytoplasmic and vacuolar compartments, respectively. The cytoplasmic pH values indicate that the tissue was adequately supplied with oxygen (Fox & Ratcliffe, 1990).

The sizes of the P pools in the roots were estimated in a similar way as for the hyphae. The amounts of polyP and vacuolar Pi were more similar to each other in the mycorrhizal roots than in external hyphae, and corresponded to the level of vacuolar Pi in the external hyphae. Despite the variation seen in Figs 6, 7 and 8, some trends could be observed. PolyP increased to a constant level after additional P supply in both treatments 1 and 2 (Figs 6 and 7), while roots from treatment 3 contained polyP at this level from the first harvest (Fig. 8). Amounts of vacuolar Pi increased with additional P supply in both treatments 1 and 2, but the variation was high (Figs 6 and 7). The mycorrhizal roots from treatment 3 contained very low amounts of vacuolar Pi (Fig. 8). Some of the variation in signal areas corresponded to the variation in quantity of total hyphae (see also Table 1), especially in the case of high amounts of hyphae and large signals in the spectra.

Figure 7.

In vivo31P nuclear magnetic resonance (NMR) spectra of mycorrhizal cucumber (Cucumis sativus) roots from sequentially harvested pots, which had received 100 mg P per pot at 0 h and a nutrient solution with 0.7 mm P during the previous 2 wk (treatment 2). Fresh weight of samples in the NMR tube varied between 0.12 g and 0.40 g and did not include all harvested material. Spectra were calibrated against a reference signal. Peak assignments were as follows: a, several phosphomonoesters; b, cytoplasmic inorganic orthophosphate (Pi); c, vacuolar Pi; d, γ-NTP; e, β-NDP/terminal polyphosphate (polyP) residues; f, α-NTP; g, uridine diphosphoglucose; h, β-NTP; i, penultimate polyP residues; j, central polyP residues.

Average chain length in external hyphae and mycorrhizal roots

The average chain-length of the NMR-visible polyP can be estimated from the ratios of the areas of the signals in the spectra for terminal, penultimate and central Pi residues in the polyP chain. Lengths were estimated for both external hyphae and mycorrhizal roots using the formula 2(Pter + Ppen + Pcen)/Pter, where Pter, Ppen and Pcen represent the areas of the signals for the terminal, penultimate and central Pi residues in the polyP chain. Signals for terminal and penultimate Pi residues in the polyP chain overlapped with the signals from phosphates of NTP in the spectra of mycorrhizal roots. However, the areas needed for the estimation of the average polyP chain length were obtained by calibrating the overlapping signals with the detached signal of α-NTP. An average chain length of 9–24 Pi residues (mean = 14) was estimated for hyphae of treatment 1 in the 16–96 h harvest interval. Hyphae from the 1–96 h interval of treatment 2 had average chain lengths in the range 11–21 Pi residues (mean = 14) while hyphae from treatment 3 contained 9 and 11 Pi residues at 1 h and 96 h after P supply. The corresponding estimates for average number of Pi residues in polyP of roots were 6–14 (mean = 11) and 6–9 (mean = 8) for treatments 1 and 2, respectively. In treatment 1, the first length estimate could be obtained 24 h after the supply of P. Roots from treatment 3 contained chains of 10 and 9 Pi residues at 1 h and 96 h after P supply. Overall, the average chain lengths were 13 Pi residues in the external hyphae and nine Pi residues in the mycorrhizal roots.

Further characterization of polyP

Five synthetic polyP glasses gave rise to NMR spectra containing the expected signals for polyP (spectra not shown). The average chain lengths estimated were 6 (type 5 polyP), 14 (type 15 polyP), 22 (type 25 polyP) and 34 (type 35 polyP). The chain length of type 75+ polyP could not be determined as the spectrum contained a signal only for the central polyP residues.

The amount and chain length of polyP in G. intraradices were further investigated using a combination of extraction procedures, colorimetric measurements and NMR. The absorption spectra of the metachromatic reaction of sequentially extracted polyP (see the Materials and Methods section) and toluidine blue showed that long-chain and granular polyP were present in the EDTA and PC extracts, respectively, of pooled samples of hyphae from the 10–96 h harvests of treatments 1 and 2 (Fig. 9b,c). By contrast, the EDTA and PC extracts of hyphae from the 1–5 h sampling interval contained no long-chain or granular polyP (absorption spectra not shown). Short-chain polyP could not be identified by metachromasy in any of the TCA fractions; accordingly, 31P NMR spectra of the TCA fractions contained no signals for short-chain polyP (Fig. 10a), while some Pi could be identified. However, 31P NMR spectroscopy confirmed the presence of polyP in the EDTA and PC fractions of the extracts of hyphae from treatment 2 (Fig. 10b,c). The percentages of long-chain and granular polyP in proportion to short-chain polyP in the in vivo measurements were estimated from the respective areas of signals in the 31P NMR spectra of the EDTA and PC extraction fractions, respectively (Fig. 10b,c), as proportions of areas from the in vivo31P NMR spectra (Figs 1–3). This calculation showed that only 5% long-chain and 2% granular polyP were present in hyphae from the 10–96 h harvest interval. No terminal or penultimate Pi residues were present in the spectra for the polyP in the EDTA and PC fractions (Fig. 10b,c) and this is parallel to the spectrum obtained for the synthetic type 75+ polyP (see above). Large amounts of polyP with, for example, 60–80 residues, would thus have markedly increased the estimated average chain length, which however, remained low. The presence of polyP with an even higher number of Pi residues cannot be excluded, as this would not be visible by NMR.

Figure 9.

Absorption spectra of: (a) toluidine blue in the absence and presence of 1 or 5 µg polyphosphate (polyP) standards (synthetic polyP glasses with average chain length 5, 25 and 75+); (b) toluidine blue in the absence and presence of polyP in successive trichloroacetic acid (TCA), phenol–chloroform (PC) and ethylenediaminetetraacetic acid (EDTA) extracts of Glomus intraradices hyphae pooled from the 10–96 h harvests in treatment 1 (see Table 1); (c) toluidine blue in the absence and presence of polyP in successive TCA, PC and EDTA extracts of hyphae pooled from the 10–96 h harvests in treatment 2 (see Table 1).

Figure 10.

31P NMR spectra of (a) trichloroacetic acid (TCA) (b) ethylenediaminetetraacetic acid (EDTA) and (c) phenol–chloroform (PC) fractions of successively extracted Glomus intraradices hyphae pooled from the 10–96 h harvests in treatment 2. Peak assignments are as follows: a, inorganic orthophosphate (Pi); b, central polyphosphate (polyP) residues.

Since no polyP could be found in the TCA fractions of the extracts of hyphae, the TCA component of the successive extraction procedure was tested for recovery of short-chain polyP (data not shown). Both 31P NMR and total P content confirmed that only one-third of the expected polyP could be extracted by TCA at low polyP concentration.

Discussion

The present work is the first in vivo study of the dynamics of P pools in arbuscular mycorrhizas under semi-natural conditions and it demonstrates that in vivo31P NMR spectroscopy can be applied to identify and characterize P pools in G. intraradices hyphae and their associated roots. Our work provides the first evidence for storage of polyP in the acidic vacuoles and we show that polyP is synthesized before any vacuolar Pi becomes visible.

In vivo31P NMR spectra of G. intraradices and mycorrhizal cucumber roots

It is generally the mobile, lower molecular weight metabolites that contribute to NMR spectra of living tissue (Pfeffer & Shachar-Hill, 1996). These molecules are visible only if their concentration exceeds the detection threshold and if the resonance intensity is not broadened as a result of immobilization. Our detection of only vacuolar Pi and polyP signals in G. intraradices is consistent with 31P NMR spectra acquired from a range of ectomycorrhizal fungi (Martin et al., 1983, 1985; Grellier et al., 1989; Ashford et al., 1994) and AM fungal spores (Shachar-Hill et al., 1995). Our failure to detect cytoplasmic Pi or NTP in the fungus could be caused by a small cytoplasmic volume or by concentrations of these analytes being below the detection threshold. Vacuoles are known to store Pi at much higher concentrations than the 5–10 mm that is common in cytoplasm (Klionsky et al., 1990; Smith et al., 2001). Furthermore, the vacuole can occupy a large volume, and multiple vacuoles are common in fungi (Jennings, 1995).

All expected signals for P metabolites were present in the spectra of P-treated mycorrhizal cucumber roots. Their chemical shift for vacuolar Pi was almost identical to that of vacuolar Pi in external mycelium, so it was not possible to distinguish between root and fungal vacuolar Pi. However, the absence of polyP in spectra of nonmycorrhizal roots (Rasmussen et al., 2000) confirmed its fungal origin.

Pools and dynamics of P in G. intraradices and mycorrhizal cucumber roots

PolyP signals were detectable in spectra of P-starved hyphae 5 h after P supply (well before other P signals appeared in hyphae and roots). In a previous experiment with Scutellospora calospora, P uptake into polyP could be detected in hyphae as early as 0.5 h after the supply of P to a P-starved mycelium (unpubl. data). This is in agreement with Ezawa et al. (2004) who detected a rather high rate of polyP accumulation in external hyphae of Archeospora leptoticha 1 h after supplying additional P to the growth medium. Such uptake rates are much faster than those of G. intraradices in monoxenic cultures where 32Pi could be detected in the hyphae at 14 h but not at 7 h after 32Pi supply (Nielsen et al., 2002).

The appearance of polyP before vacuolar Pi is consistent with earlier observations in yeast (Saccharomyces cerevisiae), where a range of methods were used to indicate that Pi uptake and polyP synthesis are regulated in concert (Castrol et al., 1999; Ogawa et al., 2000; and see Harold, 1966). PolyP accumulation appeared to be required to maintain a high rate of Pi uptake over the long-term (Ogawa et al., 2000) and our results indicate that a similar relationship could be operating in AM fungi. Rapid polyP synthesis clearly has advantages both for osmotic control (Mimura, 1999) and maintenance of cytoplasmic Pi concentration (Martin et al., 1994). Preliminary investigations with excised G. intraradices hyphae suggest that polyP was hydrolysed 24 h after transfer of the mycelium to P free medium (N. Viereck, unpubl. data). The appearance of polyP before vacuolar Pi is, however, contrary to results on cultures of ectomycorrhizal fungi (Martin et al., 1983, 1985; Grellier et al., 1989) where P was predominately accumulated as intracellular Pi, followed by polyP. The relative amounts of polyP varied according to growth conditions (especially P concentration), growth phase and fungal species. Germinating spores of an AM fungus also contained high levels of vacuolar Pi relative to polyP (Shachar-Hill et al., 1995), which may reflect differences in P metabolism between growth phases of AM fungi. Our results established that the polyP pool in external G. intraradices hyphae was equal to or considerably larger than the pool of Pi, and is therefore the main pool of soluble P metabolites in the mycelium. This suggests that polyP has an important role in fungal Pi accumulation. In ectomycorrhizal mycelium, polyP was only a fraction of the total P (3–17%), and similar to the Pi fraction (14–17%) of total P. The remaining P was immobilized and NMR-invisible (Martin et al., 1983). Similar amounts of polyP have been detected in AM fungi (Capaccio & Callow, 1982; Solaiman et al., 1999), whereas yeast accumulated larger amounts of polyP, comprising 37% of the total cellular P (Ogawa et al., 2000).

A substantial proportion of the NMR-visible P in mycorrhizal roots was of fungal origin, as estimated from the relative areas of the polyP and vacuolar Pi signals. Assuming that the fungus occupies at least one-tenth of these heavily colonized roots (Hepper, 1977), the intraradical mycelium contained polyP levels that were similar to those of the external mycelium.

Characterization of polyP and consequences for P translocation

The presence of polyP at low P supply and its location in vacuoles is consistent with a role for polyP in the transport of Pi from soil to host root. PolyP translocation could occur immediately after P becomes available, via transport in the motile, pleiomorphic system of interlinked P-rich tubular vacuoles (see the Introduction and Ashford, 1998; Allaway & Ashford, 2001).

The observation of primarily short-chain length polyP in the hyphae (< 20 Pi residues), independent of P treatment, agrees with previous reports of polyP average chain length in mycorrhizas (Martin et al., 1985; Ashford et al., 1994; Rasmussen et al., 2000). PolyP granules have been reported in preparations of mycorrhizas (Cox et al., 1975; Ashford et al., 1985; Solaiman et al., 1999), but their occurrence in vivo and their role in long distance transport in a vacuolar system have been questioned (Cox & Tinker, 1976; Orlovich & Ashford, 1993; Ashford, 1998). While in vivo NMR alone is not suitable for determination of the chain-length distribution of polyP because of the poor mobility of the long-chain polyP, X-ray microanalysis (Bücking & Heyser, 1999) and electrophoresis (Solaiman et al., 1999; Ezawa et al., 2004) have revealed the presence of polyP granules in mycorrhizal fungi and suggested a significant contribution of the long-chain polyP. In the present work, extracts of hyphae supplied with high P for more than 10 h contained only small amounts of long-chain and granular polyP, which would not have influenced the in vivo31P NMR spectra. More work using a range of methods is required for more precise determination of the chain-length distribution of polyP in mycorrhizal fungi at different growth conditions.

The slightly shorter average chain length for polyP in the mycorrhizal roots than in the external hyphae could result from hydrolytic activity (Solaiman et al., 1999; Solaiman & Saito, 2001) in accordance with the distribution of enzymes involved in polyP metabolism (Capaccio & Callow, 1982; Castrol et al., 1999; Ezawa et al., 2001). Ezawa et al. (2001) suggested dominance of acidic hydrolysing activity in intraradical hyphae, and they proposed that an acid phosphatase is responsible for polyP hydrolysis.

Detection of predominantly short-chain polyP in external mycelium of G. intraradices by in vivo31P NMR was not confirmed by successive extractions, and our results indicated some limitations in extraction of TCA-soluble short-chain polyP. Neither the absorption spectra nor the 31P NMR spectra of the TCA fractions indicated short-chain polyP and only one-third of the short-chain polyP could be recovered by TCA at low polyP concentration. Moreover, we observed no marked increase in the Pi signal in the NMR spectra, which would have resulted from acid hydrolysis of polyP. This could partly explain why no short-chain polyP could be extracted in the present work, but further investigation is required to assess the effectiveness of TCA in extraction of short chain polyP.

In conclusion, we have demonstrated the usefulness of in vivo NMR for dynamic studies of P metabolism in AM fungi, using small amounts of material. In future work we will need to standardize the amount of material inside the detection volume to make the method more quantitative. Our combined study of NMR and sequential extraction indicates that results obtained with the latter technique needs to be treated with caution.

Acknowledgements

The financial support by the Danish National Research Foundation is gratefully acknowledged. We thank Anne Olsen, Anette Olsen (Risø National Laboratory), Anette Christensen and Rita Buch (Roskilde University) for technical assistance and Professor Sally E. Smith (University of Adelaide) for inspiring discussions as well as for comments and suggestions to the manuscript.

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