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Keywords:

  • arbuscular mycorrhizal fungi (AMF);
  • inorganic phosphate (Pi);
  • polyphosphate (poly P);
  • polyphosphate kinase (PPK)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process.
  • • 
    Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2–8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase.
  • • 
    The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots.
  • • 
    These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that cannot reproduce themselves without colonizing roots. Various functions have been demonstrated for AMF, such as enhancing host nutrient uptake (especially inorganic phosphate, Pi); increasing host resistance to environmental stresses (e.g. drought) or to plant pathogens; forming nutritional connections between different kinds of host in natural grassland ecosystems; and contributing to the maintenance of diversity (Smith & Read, 1997). Therefore exploitation of effective inocula of the fungi has been widely expected (Saito & Marumoto, 2002).

Many pot experiments have shown that colonization by an AMF enhanced the host's Pi uptake, but quantitative evaluation of the AMF's contribution is difficult, especially in natural ecosystems. In the case of nitrogen fixation by Rhizobium spp., the contribution of the bacterium to the host's N acquisition can be evaluated by 15N labelling because the plant does not fix N by itself. However, because AMF and roots both obtain Pi from the same source, specific labelling of Pi cannot distinguish between Pi obtained directly from the soil and that obtained from the AMF. The separation of roots and hyphae into different compartments by using, for example, nylon mesh sheets, or the use of transformed root colonized by AMF, provides an excellent system for assaying AMF activity (Joner & Leyval, 1997; Smith et al., 2003), but even in this system the root compartment contains many hyphae, and quantitative evaluation of the AMF's contribution requires precise determination of the distribution of active hyphae among the compartments.

It has been suggested that AMF convert Pi taken up from the soil into inorganic polyphosphate (poly P) (Callow et al., 1978), a linear polymer of three Pi to thousands of Pi residues connected by high-energy phospho-anhydrate bonds. Poly P is a ubiquitous molecule, and all organisms investigated so far have been found to contain it. AMF have been shown to accumulate poly P in their hyphae (Cox et al., 1980), and to synthesize poly P very rapidly (Ezawa et al., 2003). Poly P has various biological functions (Kornberg et al., 1999), and the biological function of poly P in AMF remains to be clarified. Ezawa et al. (2001) showed that the activity of poly P-glucokinase in AMF was very low, and thus proposed that poly P did not function as a major Pi donor in glucose metabolism in AMF cells. It has been hypothesized that AMF translocate poly P from extraradical to intraradical mycelium (Cox et al., 1980), and depolymerize poly P before supplying Pi to the host plant cells (Solaiman & Saito, 2001). Alkaline phosphatase (ALP) has been considered a key enzyme in determining an AMF's ability to supply Pi, and the ALP activity was induced under high levels of environmental Pi (van Aarle et al., 2002; Olsson et al., 2002). However, transcription of the ALP gene was not induced by environmental Pi (Aono et al., 2004). The ALP expressed specifically in mycorrhizal roots was found to be of fungal origin (Kojima et al., 1998) and has been partially purified (Kojima et al., 2001). Although the involvement of ALP in the metabolism of poly P was questioned because Ba2+-sensitive fungal ALP did not hydrolyse poly P (Ezawa et al., 1999), the enzyme was shown to be at least partially involved in Pi efflux from isolated intraradical hyphae of AMF (Kojima & Saito, 2004).

Until recently, microquantification of poly P was difficult. Commonly used quantification methods, including staining with Toluidine blue O, DAPI or Fura-2, and determination of Pi after acid hydrolysis of poly P, are not highly specific to poly P, nor are they sensitive enough to quantify very small amounts of poly P. An enzymatic assay, in which poly P is converted into ATP by Escherichia coli polyphosphate kinase (PPK) followed by ATP quantification using the luciferin–luciferase system (Ault-Richéet al., 1998), has overcome this difficulty. Application of this technique to AMF has been successful (Ezawa et al., 2003), and it is now possible to measure poly P in small fungal samples (mg levels). Thorough investigation of the poly P dynamics in AMF would help us understand the metabolism and function of poly P in these fungi.

In the present study, we monitored changes in the poly P content of mycorrhizal roots during the colonization process. Because we first aimed to determine the poly P levels in various colonization stages, we periodically sampled mycorrhizal plants to obtain roots with various levels of colonization. We analysed the relationships between poly P content and colonization, host P status, and growth of both host and fungus.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Soil and plants

Seeds of onion plants (Allium cepa L. cv. Sensyuchukou) were purchased from Kaneko Seed Co. (Maebashi, Japan). They were surface sterilized for 30 min in hypochlorous acid with an effective chlorine concentration of ≈ 0.3%, then germinated for 1 wk on wet filter paper. The culture medium was prepared by mixing sand, agricultural soil and horticultural medium. The sand was washed with tap water to remove fine particles, and air-dried. Agricultural soil was collected from the plough layer of an experimental field at the National Institute of Livestock and Grassland Science (Tochigi, Japan), and sieved through a 2 mm mesh after air-drying. Horticultural medium was purchased from Kureha Chemical Industry Co., Ltd (Tokyo, Japan). All components of the culture medium were sterilized by autoclaving at 121°C for 20 min.

Experimental design

Onion seedlings were transplanted into 150 ml pots with or without simultaneous inoculation with spores of Gigaspora margarita (MAFF 520054) as described previously (Kojima et al., 1998). The inoculation consisted of either 10 or 50 spores per pot. There were thus three treatments: nonmycorrhizal plants (NM); plants inoculated with 10 spores (10S); and plants inoculated with 50 spores (50S). The culture medium used was a mixture of sand, agricultural soil and horticultural medium at a volume ratio of 5 : 4 : 1, and was supplemented with 1 g l−1 CaCO3. Superphosphate was added to the medium for NM plants at 2.94 g l−1 because onion requires high amounts of Pi for growth, and cannot grow well on a low-Pi medium without colonization by AMF. The Pi concentrations of fertilized and nonfertilized media, as determined by the Truog method (Japanese Society of Soil Science and Plant Nutrition, 1997), were 33 and 10 mg P kg−1 dry soil, respectively. The pH (H2O) of fertilized and nonfertilized media were 6.0 and 6.4, respectively. Twenty pots planted with two seedlings each were prepared for each treatment, and eight pots were thinned to one seedling 1 wk after transplanting. Pots with two seedlings (12 pots per treatment) were used for sampling in the early stages to obtain enough material for later analysis, and pots containing one seedling were used for later sampling. Plants were grown in a growth chamber (Koitotron 3HN-35DA, Koito Industries Ltd, Japan) with a 14 h day at 25°C and a 10 h night at 20°C.

Growth of the plants was monitored by measuring their shoot length. After growing for the period indicated, one pot from each treatment was processed as follows: the plants were harvested, separated into shoots and roots, and AMF spores were collected from the soil by the wet sieving method (Brundrett et al., 1996). The shoot samples were dried in a constant-temperature oven (DK810, Yamato, Tokyo, Japan) at 70°C for 2–3 d, ground with a mortar and pestle, and stored in a desiccator. Root samples were washed with tap water, then chopped into ≈ 1 cm pieces. An aliquot of each root sample was stained with Trypan blue to assess the degree of colonization as described previously (Brundrett et al., 1996). Other aliquots were saved for the determination of total phosphorus (total P) after drying, as above, or were stored at −80°C for poly P quantification.

Colonization rate and colonization strength were determined by the intersection method (Brundrett et al., 1996). Each intersection was classified into one of five categories according to its colonization intensity: (A) no colonization; (B) very weak colonization, only a few hyphae visible; (C) moderate colonization, less than half of the root occupied by hyphae; (D) better colonized, more than half of the root occupied by hyphae; (E) vigorous colonization, the entire root packed with hyphae. On the basis of the observed numbers of the points in each category (a–e, respectively), the colonization rate and colonization strength were calculated as follows:

  • Colonization rate = (b + c + d + e)/(a + b + c + d + e)
  • Colonization strength = (0.02b + 0.30c + 0.75d + 1.00e)/(a + b + c + d + e)

Extraction, purification and quantification of polyphosphate

About 10 mg (f. wt) of root samples, 200 mg acid-washed glass beads (1 mm in diameter), and 500 µl acetone prechilled at −20°C were mixed in a 2 ml screw-cap tube and stored on dry ice. The roots were subsequently disrupted using a Mini-Bead Beater (Biospec Products Inc., Batlesville, OK, USA) at 5000 rpm for 10 s. Disruption was repeated three times with an interval of at least 1 min on dry ice between disruptions. After removal of the acetone in a rotary evaporator (Centrifugal Concentrator CC-181 with Low Temperature Trap Unit TU-040, Tomy, Tokyo, Japan), the resulting powder was extracted with 500 µl extraction buffer (8 m urea, 50 mm Tris–Cl at pH 8.0). The mixture was centrifuged at 23 000 g for 1 min at 4°C, and the supernatant was pooled to serve as the root extract. The urea in the extracts was removed using a gel filtration spin column. For this purpose, an empty BioSpin column (Bio-Rad, Hercules, CA, USA) was packed with 0.75 ml (wet volume) of hydrated (with 10 mm Tris–Cl pH 7.5) and degassed Bio-Rad BioGel P-2 or P-6 gel, whose molecular weight cut-off value (MWCO) is 2 or 6 kDa, respectively. After removal of the hydration buffer from the gel by centrifugation at 1000g for 2 min at room temperature, 50 µl of the root extract was applied to the centre of the drained gel. The columns were then centrifuged again at 1000g for 4 min at room temperature, and the eluate was saved as the purified extract.

In a preliminary experiment, the disruption of roots in the buffer without the denaturing reagent was also examined. In this case, ice-cold 10 mm Tris–Cl (pH 7.5) was used instead of chilled acetone, and the sample was kept on ice during the interval of the disruption. The supernatant of the extract was directly used for PPK assay.

Poly P concentration was determined by the reverse reaction of PPK, as described previously (Ault-Richéet al., 1998; Ezawa et al., 2003). Escherichia coli PPK was purified from an E. coli culture that overexpresses PPK, as described elsewhere (Ezawa et al., 2003; Ohtomo et al., 2004). Purified extract (2 µl) was mixed with a final 20 µl reaction mixture consisting of 50 mm (NH4)2SO4, 4 mm MgCl2, 8 µm ADP, 40 mm HEPES–KOH (pH 7.5) and 30 U µl−1 PPK, and the solution was incubated at 37°C for 1 h, followed by heating at 95°C for 2 min to inactivate the PPK. The reacted solution was diluted 1 : 100 with ATP dilution buffer (10 mm EDTA and 100 mm Tris–Cl pH 7.5), and 20 µl of the dilution was mixed with an equal volume of CLSII reaction mixture (Roche Diagnostics, Basel, Switzerland). Chemiluminescence was measured with the Luminescencer PSN (Atto, Tokyo, Japan) as the total luminescence count in 10 s, using 0.5–500 nm ATP in ATP dilution buffer as standard. The amount of poly P is given in terms of Pi residues. The protein concentration of the root extract was determined with a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, Rockford, IL, USA) following the manufacturer's instructions, using 0–500 µg ml−1 bovine serum albumin in the extraction buffer as standard.

Determination of total P and free phosphate content of plant samples

Total P and free inorganic phosphate (free Pi) contents were determined by the molybdenum-blue method (Chen et al., 1956; Ohtomo et al., 2004). For the total P measurement, ≈ 1 mg (d. wt) ground root samples (corresponding to ≈ 10 mg f. wt) were mixed with 200 µl 30% (w/v) H2O2 and 50 µl H2SO4, and hydrolysed by heating at 200°C for 30 min or more. If a brown colour developed during heating, samples were supplemented with another 200 µl 30% H2O2 and heated further. The colourless solution generated by complete hydrolysis was diluted with distilled water to 2.25 g, and 100 µl aliquots of the diluted samples were mixed with 50 µl H2O and 50 µl of reaction solution [a mixture of 2.52% (w/v) ammonium molybdate in 1 m H2SO4, 10% ascorbic acid, and water at a 2 : 2 : 1 ratio] in a well of a 96-well microtitre plate. After incubation at 45°C for 20 min, absorbance at 820 nm (A820) was measured by using a Multiskan Ascent system (Thermo Labsystems, Helsinki, Finland). Pi concentration was determined using 10–500 nm KH2PO4 as standard.

The free Pi in shoots was extracted with distilled water. We first tried the boiling water extraction method described by Bollons & Barraclough (1997), but found that the extraction efficiency with room-temperature water was close to that with boiling water. Ground shoot samples (2 mg) were mixed with 400 µl distilled water by vortexing. After centrifuging the solution at 23 000g for 5 min, the supernatant was saved. We then mixed 50 µl aliquots of the extracts with 100 µl 0.4 m H2SO4 and 50 µl of reaction solution [a mixture of 2.52% (w/v) ammonium molybdate in 1 m H2SO4, 10% ascorbic acid and water at a 2 : 2 : 1 ratio] in a well of a 96-well microtitre plate, and A820 was measured as described above.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Improvement of polyphosphate extraction and purification methods from AMF samples

Our preliminary results indicated that disruption of the hyphae using the Mini-Bead Beater in 10 mm Tris–Cl (pH 7.5) as an extraction buffer was not effective for extracting poly P from this fungus. When we supplemented the disruption buffer with a high concentration of protein-denaturing reagent such as guanidine isothiocyanate or urea, we were able to extract much more poly P, and 8 m urea proved to be the most effective extracting agent that we tested (data not shown). Table 1 shows the poly P content in various mycorrhizal samples. In the extraradical hyphae or spores, the poly P contents after gel filtration through BioGel P-2 and P-6 were similar. In contrast, extracts from mycorrhizal roots showed a substantially lower poly P content when passed through the P-6 gel than through the P-2 gel. On this basis, we used BioGel P-2 thereafter for the purification of poly P from mycorrhizal samples.

Table 1.  Polyphosphate (poly P) in mycorrhizal roots, in spores and in hyphae
SampleWeeksProtein (mg g−1 f. wt)Poly P/protein (nmol mg−1) P-6/P-2 (%)
P-2P-6
  • Values are averages of at least three independent measurements (± SD).

Spores 74 (± 5) 409 (± 24) 356 (± 33) 87
Extraradical hyphae610 (± 4)1391 (± 26)1442 (± 31)104
 914 (± 2) 625 (± 169) 721 (± 178)115
Mycorrhizal roots612 (± 1)  23 (± 13)   4 (± 3) 18
927 (± 18)  16 (± 5)  10 (± 5) 64

Growth of host plant and fungus

Nonmycorrhizal, fertilized plants (NM) showed the best growth within the first 30 d after transplanting (Fig. 1). Increases in growth rates for the 50S and 10S treatments were observed ≈ 20 and 30 d, respectively, after transplantation. Final shoot length of 50S plants was greater than that of NM plants, and the shoot length of 10S plants were almost comparable with that of NM at the end of the experiment. In the inoculated treatments, the colonized root weight increased with time (Fig. 2a). Colonized root weight in the 50S plants increased until the end of the experiment, although the colonization strength increased only slightly after 23 d from transplanting (data not shown). No hyphae were observed in the roots of NM plants. In the inoculated treatments, the number of spores started to increase after 30 d from transplanting (Fig. 2b). The degree of the increase (number of spores observed after harvesting divided by the number of spores inoculated, which was either 10 or 50) was larger for the 10S plants.

image

Figure 1. Growth of the host plant (onion, Allium cepa) during the experiment as indicated by shoot length. NM, 50S and 10S represent noninoculated, fertilized treatment; inoculated treatment (50 spores per pot); and inoculated treatment (10 spores per pot), respectively. Error bars, SD within each treatment. Open circles, NM; filled circles, 50S; squares, 10S; triangles, sampling.

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image

Figure 2. Growth of the mycorrhizal fungus during the experiments as assessed by colonized root weight (a) and number of spores recovered from the soil (b). 50S and 10S represent plants (onion, Allium cepa) inoculated with 50 or 10 spores per pot, respectively. No significant colonization and no spores were observed in noninoculated plants (NM, nonmycorrhizal; data not shown). Circles, 50S; squares, 10S.

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Phosphorus balance in mycorrhizal plants

Total P uptake by the host plant increased over time, and the rate of increase was greatest in 50S plants (Fig. 3a). The difference between uptake by plants in the NM (fertilized) treatment and the 50S (nonfertilized) treatment was first observed ≈ 30 d after transplanting. The total P content in the 10S treatment also increased 35 d after transplanting, and became higher than in the NM treatment 45 d after the transplanting. P uptake was promoted by mycorrhizal colonization, because no significant difference in total root weight was observed among the series (data not shown). The free Pi content in the shoots also increased as the plants grew (Fig. 3b), showing a similar changing pattern as observed for total P.

image

Figure 3. Phosphorus status in host plants (onion, Allium cepa) during the experiment including total P content (a), and free inorganic phosphate (Pi) content in shoots (b). NM, 50S and 10S represent noninoculated, fertilized treatment; treatment with 50 spores per pot; and treatment with 10 spores per pot, respectively. Open circles, NM; filled circles, 50S; squares, 10S.

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Polyphosphate content in roots colonized by AMF

The poly P content in mycorrhizal roots increased as colonization proceeded, but accumulation of poly P was not observed in the roots of NM (data not shown). In the 50S treatment, the 17 d samples (from the second sampling) contained a significant concentration of poly P (in terms of nmol Pi residues mg−1 protein). The concentration increased until 35–36 d, then a gradual decrease occurred thereafter (Fig. 4a). Total poly P amount per plant in the 50S treatment showed a sharp increase until 35–40 d after transplanting, and the rate slowed down thereafter (Fig. 4b). In the 10S treatment, the increase in poly P concentration and the accumulation of poly P were retarded almost 10 d compared with the 50S treatment (Fig. 4).

image

Figure 4. Accumulation of poly P in mycorrhizal roots during colonization as shown by polyphosphate (poly P) concentration (a) and total poly P content (b) per plant. 50S and 10S represent plants (onion, Allium cepa) inoculated with 50 or 10 spores per pot, respectively. Essentially no poly P was detected in the roots without mycorrhizal colonization (NM, nonmycorrhizal; data not shown). Circles, 50S; squares, 10S.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Quantification of polyphosphate in mycorrhizal samples

We successfully adapted the method for enzymatic quantification of poly P to various AMF samples, including spores, extraradical hyphae and plant roots colonized by AMF. In the extraction of poly P from these samples, the mechanical disruption in nondenaturing buffer, which has been used with the yeast Saccharomyces cerevisiae (Ogawa et al., 2000), gave a significantly lower value than expected, presumably because some basic components from the cell bound to the poly P and inhibited its recovery in the extract. Supplementing the extraction buffer with a high concentration of denaturing reagent greatly improved the recovery of poly P, although we found that the extracted poly P was less stable in the presence of these reagents, especially at room temperature. In the end, we adapted the extraction method as follows. After mechanically disrupting the samples in cold acetone, the acetone was removed by evaporation, then the resultant dry powder was extracted with a buffer containing 8 m urea. After the extraction, the urea and other compounds with low molecular weight, including ATP and ADP from fungal or plant cells, were removed by simple gel filtration. We avoided the previously reported glassmilk purification technique (Ault-Richéet al., 1998), because it has been reported that this procedure fails to recover poly P shorter than 60 Pi residues (Ogawa et al., 2000). Very short poly P might be lost during the gel filtration step, and the expected chain length of unrecovered poly P was ≈ 20 Pi residues when the MWCO of the gel filtration medium was 2 kDa. However, because the PPK reverse reaction showed low reactivity against poly P shorter than 20 Pi residues (Ohtomo et al., 2004), the loss of poly P shorter than 20 Pi residues by gel filtration would not affect the poly P quantification with the present method using PPK.

The concentration of poly P in the extraradical hyphae of G. margarita in this study was 271 µg P g−1 f. wt (1391 nmol P mg−1 protein × 10 mg protein g−1 f. wt in the hyphae 6 wk after transplanting; Table 1), comparable with the concentrations reported by previous studies. Solaiman et al. (1999) reported that the extraradical hyphae of G. margarita contained 200–300 µg P g−1 f. wt poly P. Ezawa et al. (2003) reported an even higher value (a maximum of 12 000 nmol P mg−1 protein) in the marigold (Calendula officinalis)–Archaeospora leptoticha system, which might be caused by a different host–fungus combination or the effect of poly P overplus (Harold, 1966; Pestov et al., 2004). These poly P contents of AMF were higher than those observed in other organisms: 60–120 nmol P mg−1 protein was reported in E. coli (Ault-Richéet al., 1998; Kuroda et al., 1999), and 300 nmol P mg−1 protein in S. cerevisiae (Sethuraman et al., 2001). However, they were comparable with levels reported for other organisms that are known to accumulate poly P in high concentrations: 700 nmol P mg−1 protein in Klebsiella aerogenes (Ohtake et al., 1999); and 7000 nmol mg−1 protein in Chlamydomonas acidophila (K. Nishikawa and coworkers, pers. comm.). The AMF in the present study is thus confirmed to be an accumulator of poly P.

The poly P level in mycorrhizal roots was 8.6 mg P g−1 f. wt (23 nmol mg−1 protein × 12 mg g−1 protein f. wt; Table 1). This corresponds to 330 µg P g−1 intraradical hyphae, if the mycorrhizal roots are assumed to contain 26 mg hyphae g−1 f. wt, as measured by Solaiman et al. (1999) for the same host–fungus combination as in the present study. This estimate is also consistent with the value (340 µg P g−1 hyphae) obtained by Solaiman et al. (1999), and higher than the value (3.9 µg P g−1 root f. wt) reported by Callow et al. (1978) using the onion–Glomus mosseae system. Nonmycorrhizal onion roots contained no detectable poly P, as reported by Callow et al. (1978). Nuclear magnetic resonance spectroscopy (NMR) analyses showed that mycorrhizas contained short-chain poly P with a chain length < 20 Pi residues (Rasmussen et al., 2000; Viereck et al., 2004). However, they did not quantify the total poly P content in mycorrhizal roots. Furthermore, NMR is not able to detect insoluble or immobile poly P (Chen, 1999). As discussed above, the short-chain poly P was not detected by the present method. So it remains to be determined in further studies how much of the total poly P in AMF and mycorrhizal roots is detectable or undetectable by the PPK method. It is also noteworthy that different fungi might show different poly P accumulation patterns. Boddington & Dodd (1999) found that Gigaspora rosea accumulated large amounts of DAPI-stainable poly P in the extraradical hyphae, although Glomus manihotis did not.

Poly P pool in mycorrhizal roots as phosphate supply to host

Poly P in mycorrhizal roots could pass through the BioGel P-2 gel filtration medium (whose MWCO is 2 kDa) but not through the P-6 medium (whose MWCO is 6 kDa), although the poly P from extraradical hyphae and spores could pass through both media (Table 1). This indicates that the poly P in mycorrhizal roots was shorter than that in the spores and the extraradical hyphae. This was consistent with the result obtained by Solaiman et al. (1999), and strongly supports the model that AMF depolymerize poly P in the root to supply Pi to the host plant. Viereck et al. (2004) reported that the average chain length of NMR-visible poly P in intraradical hyphae of Glomus intraradices colonizing cucumber was around nine Pi residues and shorter than the ≈ 13 residues in the extraradical hyphae. This may be in line with the above finding.

The data in the present study indicated that the poly P content of roots increased as mycorrhizal colonization proceeded. As shown in Fig. 5, we found that the weight of colonized roots and the poly P content of the mycorrhizal roots were highly correlated, with a correlation coefficient of 0.94 in the current experimental system (G. margarita–onion). The regression equation indicated that the cube root of the poly P content was proportional to the square root of the weight of colonized roots (Fig. 5). This is reasonable because the intraradical hyphae and poly P are both distributed three-dimensionally within the host root, although colonization was evaluated using two-dimensional surveillance under the microscope. A high correlation between poly P amount per plant and colonized root weight per plant also supports the model that AMF provides Pi to the host via the poly P pool.

image

Figure 5. Correlation between mycorrhizal colonization and polyphosphate (poly P) content. 50S and 10S represent plants (onion, Allium cepa) inoculated with 50 and 10 spores per pot, respectively. Circles, 50S; squares, 10S.

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Based on the observation that poly P content showed a slow or almost no increase 40 d after transplanting (Fig. 4b) while the weight of colonized roots kept increasing (Fig. 2a), we hypothesized that the poly P content in the mycorrhizal root reflects the biological activity of AMF. We assume that the Pi-supplying activity of the AMF might slow down during this phase because the concentration of poly P in mycorrhizal roots decreased (Fig. 4a), although the colonization strength remained almost constant (data not shown). The comparison of 6- and 9-wk samples in Table 1 suggests that the average chain length of poly P in mycorrhizal root increased in the later growing phase. This might be a result of a slowing of depolymerization of poly P, suggesting a decline in Pi-supplying activity from the poly P pool in the later growing phase. Thus poly P can provide information different from colonization rate, and may be a more suitable measure than colonization for evaluating the Pi-supplying activity of AMF.

In conclusion, our results strongly support the hypothesis that the AMF supplies Pi to the host via the pool of poly P, because (i) poly P in the AMF was shorter in the mycorrhizal root; and (ii) poly P contents in mycorrhizal roots were highly correlated with mycorrhizal colonization, especially in the early colonization stage during which most of the fungus in the root would maintain its biological activity. In the later colonization phase, the Pi-supplying activity of AMF might decrease, and that might be a reason for the decrease in poly P concentration or the increase in poly P chain length in mycorrhizal roots. This further suggests that poly P could serve as a molecular indicator for the ability of AMF to supply Pi to the host plant.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by the funds for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of the Bio-oriented Technology Research Advancement Institution, Japan. The authors are grateful to Profesor R. Koide for his critical reading of this manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Van Aarle IM, Rouhier H, Saito M. 2002. Phosphate activities of arbuscular mycorrhizal intraradical and extraradical mycelium, and their relation to phosphorus availability. Mycological Research 106: 12241229.
  • Aono T, Maldonado-Mendoza IE, Dewbre GR, Harrison MJ, Saito M. 2004. Expression of alkaline phosphatase genes in arbuscular mycorrhizas. New Phytologist 162: 525534.
  • Ault-Riché D, Fraley C, Tzeng C, Kornberg A. 1998. Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli. Journal of Bacteriology 180: 18411847.
  • Boddington CL, Dodd JC. 1999. Evidence that differences in phosphate metabolism in mycorrhizas formed by species of Glomus and Gigaspora might be related to their life-cycle strategies. New Phytologist 142: 531538.
  • Bollons HM, Barraclough PB. 1997. Inorganic orthophosphate for diagnosing the phosphorus status of wheat plants. Journal of Plant Nutrition 20: 641655.
  • Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N. 1996. Working with Mycorrhizas in Forestry and Agriculture. Canberra, Australia: Australian Center for Agricultural Research.
  • Callow JA, Capaccio LCM, Parish G, Tinker PB. 1978. Detection and estimation of polyphosphate in vesicular–arbuscular mycorrhizas. New Phytologist 80: 125134.
  • Chen KY. 1999. Study of polyphosphate metabolism in intact cells by 31-P nuclear magnetic resonance spectroscopy. In: SchröderHC, MüllerWEG, eds. Inorganic Polyphosphates. Berlin: Springer, 253271.
  • Chen PS, Toribara TY, Warner H. 1956. Microdetermination of phosphorus. Analytical Chemistry 28: 17561758.
  • Cox G, Moran KJ, Sanders F, Nockolds C, Tinker PB. 1980. Translocation and transfer of nutrients in vesicular–arbuscular mycorrhizas. III. Polyphosphate granules and phosphorus translocation. New Phytologist 84: 649659.
  • Ezawa T, Kuwahara S, Sakamoto K, Yoshida T, Saito M. 1999. Specific inhibitor and substrate specificity of alkaline phosphatase expressed in the symbiotic phase of the arbuscular mycorrhizal fungus, Glomus etunicatum. Mycologia 91: 636641.
  • Ezawa T, Smith SE, Smith FA. 2001. Enzyme activity involved in glucose phosphorylation in two arbuscular mycorrhizal fungi: indication that polyP is not the main phosphagen. Soil Biology and Biochemistry 33: 12791281.
  • Ezawa T, Cavagnaro TR, Smith SE, Smith FA, Ohtomo R. 2003. Rapid accumulation of polyphosphate in extraradical hyphae of an arbuscular mycorrhizal fungus as revealed by histochemistry and a polyphosphate kinase/luciferase system. New Phytologist 161: 387392.
  • Harold FM. 1966. Inorganic polyphosphate in biology: structure, metabolism, and function. Bacteriological Reviews 30: 772794.
  • Japanese Society of Soil Science and Plant Nutrition. 1997. Methods for Analysis of Soil Environment. Tokyo: Hakuyusha, 267269 (in Japanese).
  • Joner EJ, Leyval C. 1997. Uptake of 109Cd by roots and hyphae of a Glomus mosseae/Trifolium subterraneum mycorrhiza from soil amended with high and low concentrations of cadmium. New Phytologist 135: 353360.
  • Kojima T, Saito M. 2004. Possible involvement of hyphal phosphatase in phosphate efflux from intraradical hyphae isolated from mycorrhizal roots colonized by Gigaspora margarita. Mycological Research 108: 610615.
  • Kojima T, Hayatsu M, Saito M. 1998. Intraradical hyphae phosphatase of the arbuscular mycorrhizal fungus, Gigaspora margarita. Biology and Fertility of Soils 26: 331335.
  • Kojima T, Hayatsu M, Saito M. 2001. Electrophoretic detection and partial purification of the phosphatase specific for arbuscular mycorrhizal symbiosis. Bulletin of the National Grassland Research Institute 60: 918 (in Japanese with English abstract).
  • Kornberg A, Rao N, Ault-Riché D. 1999. Inorganic polyphosphate: a molecule of many functions. Annual Review of Biochemistry 68: 89125.
  • Kuroda A, Tanaka S, Ikeda T, Kato J, Ohtake H. 1999. Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli. Proceedings of the National Academy of Sciences, USA 96: 1426414269.
  • Ogawa N, DeRisi J, Brown P. 2000. New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis. Molecular Biology of the Cell 11: 43094321.
  • Ohtake H, Kuroda A, Kato J, Ikeda T. 1999. Genetic improvement of bacteria for enhanced biological removal of phosphate from wastewater. In: SchröderHC, MüllerWEG, eds. Inorganic Polyphosphates. Berlin: Springer, 299311.
  • Ohtomo R, Sekiguchi Y, Mimura T, Saito M, Ezawa T. 2004. Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography. Analytical Biochemistry 328: 139146.
  • Olsson PA, Van Aarle IM, Allaway WG, Ashford AE, Rouhier H. 2002. Phosphorus effects on metabolic process in monoxenic arbuscular mycorrhiza cultures. Plant Physiology 130: 11621171.
  • Pestov NA, Kulakoxskaya TV, Kulaev IS. 2004. Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess. FEMS Yeast Research 4: 643648.
  • Rasmussen N, Lloyd DC, Ratcliffe RG, Hansen PE, Jakobsen I. 2000. 31 P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi. Plant and Soil 226: 245253.
  • Saito M, Marumoto T. 2002. Inoculation with arbuscular mycorrhizal fungi: the status quo in Japan and the future prospects. Plant and Soil 244: 273279.
  • Sethuraman A, Rao NN, Kornberg A. 2001. The endopolyphosphatase gene: essential in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, USA 98: 85428547.
  • Smith SE, Read DJ. 1997. Mycorrhizal Symbiosis, 2nd edn. Cambridge, UK: Academic Press.
  • Smith SE, Smith FA, Jakobsen I. 2003. Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. Plant Physiology 133: 1620.
  • Solaiman MZ, Saito M. 2001. Phosphate efflux from intraradical hyphae of Gigaspora margarita in vitro and its implication for phosphorus translocation. New Phytologist 151: 525533.
  • Solaiman MZ, Ezawa T, Kojima T, Saito M. 1999. Polyphosphate in intraradical and extraradical hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita. Applied and Environmental Microbiology 65: 56045606.
  • Viereck N, Hansen PE, Jakobsen I. 2004. Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo31P NMR spectroscopy. New Phytologist 162: 783794.