Phosphate is at the heart of nutrient exchange in mycorrhizas. Because 31P NMR gives information on the amounts, forms and locations of phosphorus metabolites it has been useful in studying P nutrition in both EC and AM systems. Martin and co-workers carried out the first 31P study of axenically cultured ectomycorrhizal fungi (Cenococcum geophilum and Hebeloma crustuliniforme) in 1983. This report demonstrated the direct observation of polyphosphate (polyP), whose central phosphates gives a distinctive peak at −22 ppm in the 31P NMR spectrum. Such spectra also show that polyP (the form in which phosphate is usually stored by fungi; Jennings, 1995) represents much of the mobile phosphorous present in the EC fungal tissue. This study also showed that polyP contained only a fraction (approx. 8%) of the total phosphate in the mycelium, most of the rest being in phospholipids, DNA and other immobile forms of P that do not contribute to such in vivo31P NMR spectra. In later studies the NMR-observable (mobile, low molecular weight) polyP was found to correspond to a minimum of 80% of the acid-extractable polyP in the mycelium of H. crustuliniforme, and in vivo31P spectroscopy revealed that under phosphorous starvation this fungus degrades polyP to Pi (Martin et al., 1985b). The average chain length of the mobile polyP can be deduced from the ratio of the sizes of the peaks of the terminal and internal phosphate peaks in 31P spectra; thus, in H. crustuliniforme this comparison yielded an average chain length of 11 phosphate residues (Martin et al., 1985b). These values were in agreement with those obtained previously from chemical analysis of polyP extracted from this fungus (Rolin et al., 1984). More recently, polyP chain lengths of a similar size (15 units/chain) have been characterized in Pisolithus tinctorius also by 31P NMR (Ashford et al., 1993).
Further information on the physical state of polyP in axenically cultured EC fungi was derived from measurements of the ‘spin-lattice’ relaxation rate for the 31P NMR signals from polyP (Martin et al., 1985b). This spectroscopic parameter can give information on the mobility of molecules in vivo and the spin lattice relaxation behaviour of polyP was consistent with the formation of ‘supramolecular aggregates’ of these short polyP chains in free-living H. crustuliniforme.
Concerning the symbiotic state of EC fungi, Loughman & Ratcliffe (1984) first demonstrated the presence of polyP, and active incorporation of Pi into mobile polyP in living mycorrhizal beech (Fagus sylvatica) root tips. Subsequently in vivo31P NMR spectra of synthetic mycorrhiza of birch (Paxillus involutus) showed that levels of polyP were somewhat higher (10–40% of the total soluble phosphate) than those of free-living mycelium of the fungus (Grellier et al., 1989). In a study of intact mycorrhizal red pine roots (Pinus resinosa ×Hebeloma arenosa) MacFall et al. (1992) showed that the Pi : polyP ratio remained the same (approximately 1.8) when plants were grown for 19 wk in either P-amended or unamended soil. These findings were interpreted as supporting the idea that the fungus, by controlling the synthesis of polyP, can modulate Pi flow within the mycorrhizal root, and thereby regulate the amounts of Pi available for transport to the shoots. Gerlitz & Werk (1994) also followed the levels of Pi and polyP by 31P NMR in mycorrhizal pine and beech roots. Their findings showed that mobile polyP (chain lengths < 100) increased by 5–10% over a period of 20 min after exposure to Pi and then returned to their initial levels. The authors suggest that during this short period of time incoming Pi is rapidly incorporated into mobile polyP, and that it then becomes NMR-invisible as the chain length increases and/or it becomes immobilized. Recently, in an in vivo31P study of chestnut (Castanea sativa) mycorrhizas Martins et al. (1999) have demonstrated the accumulation of phosphate, mostly in the form of polyP, over a period of 3 months, in contrast to nonmycorrhizal roots, which accumulated only Pi (Fig. 1). The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhizal ones, and the authors interpreted this in terms of dependence of the host on fungal polyphosphates as a major source of phosphate. The ratio of Pi : polyP in this study was not constant over time, contrasting with the findings of MacFall et al. (1992) where this ratio did not vary.
Figure 1. The use of in vivo31P-NMR for investigating P nutrition and metabolism in the ectomycorrhizal symbiosis. Spectra taken of A uncolonized control chestnut roots and B mycorrhizal chestnut roots at successive times during development: (a) 3 wk after mycorrhizal induction; (b) 1 month after mycorrhizal induction; (c) 3 months after mycorrhizal induction. The main peaks are those of intracellular orthophosphate (Pi) and the central phosphates of polyphosphate (polyP). (Reproduced from Martins et al., 1999). These spectra show the accumulation of large amounts of fungal polyphophate as the ectomycorrhiza develops and the changing balance of Pi and PolyP over time.
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Taken together 31P NMR studies of ectomycorrhizas and EC fungi provide measurements of short and long-term uptake and incorporation of phosphate into inorganic phosphate and polyP pools. The dynamics and ratios of P in different pools differ between systems and such differences as well as the fact that not all polyP is NMR-visible necessitate care when making physiological and ecological interpretations of some findings. Nevertheless by discriminating different chain lengths and by allowing real-time measurements of different P pools, NMR has provides useful data on the dynamics of P in ectomycorrhizas.
The information available from NMR spectra on the physico-chemical state of small molecules has also proven fruitful in studies of aluminium (Al) uptake by ectomycorrhizas and EC fungi. Under acid soil conditions Al can cause severe disruption in root physiology and function, including P nutrition (Haug, 1984; Pfeffer et al., 1987; Kochian & Shaff, 1991), and mycorrhizas can moderate this toxicity. In a unique study using 27Al NMR Martin et al. (1994b) demonstrated that Al is chelated by polyP within the mycelium of the EC fungus Laccaria bicolor. The presence of four different peaks in the 27Al spectrum showed that the Al3+ ion exists in at least 4 different states and that these are probably confined to the fungal vacuole. These polyP-Al complexes were found to be very resistant to remobilization, even when the mycelium was transferred to Al-free medium. Thus the sequestration of Al by polyP could serve to protect mycorrhizal plants. Subsequently, Gerlitz (1996) demonstrated using 31P NMR that an Al-adapted EC fungus (Suillus bovinus) had a greater ability for phosphate uptake, and contained mobile polyP of shorter chain length than nonadapted fungus. Mobile shorter polyP chains were suggested to complex Al, rendering the fungus and host plant resistant to this ion’s toxic effects.
By comparison with ectomycorrhizas, few 31P NMR studies have been carried out in arbuscular mycorrhizas, mostly because of the much smaller quantities of fungal tissue available from these systems. Axenically germinating spores of the AM fungus Glomus etunicatum contain polyP of quite small chain length (Shachar-Hill et al., 1995) with the 31P signals from central, penultimate and terminal phosphate residues having intensities in the ratio 1 : 2 : 2. This points to an average chain length of 5 phosphate units. Also, and in contrast to what was found for EC fungi – in which polyP dominates (see above) – asymbiotic G. etunicatum was found to contain relatively high levels of Pi, as indicated by the large peak at 2.0 ppm corresponding to an internal compartment at pH 6.9 (Fig. 2). The presence of small, mobile polyP units and of high Pi levels may reflect the active synthesis of phosphorylated metabolites in the germination stage of the AM fungal life cycle. A recent 31P study on G. etunicatum hyphae by Rasmussen et al. (2000) found a significantly lower level of Pi relative to polyP, and this may reflect changes during the development of G. etunicatum since the earlier observations were on spores with very little hyphal material present.
Figure 2. In vivo31P NMR spectrum of ungerminated Glomus etunicatum spores showing resonances from Pi and mobile polyphosphates; a, Pi; b, terminal polyphosphate residues; c, penultimate polyphosphate residues; d, central poly phosphate residues. The ratio of signal intensities of penultimate and terminal phosphates to that of the internal phosphates shows a short average chain length of the PolyP. The Pi peak is also higher than has been observed in the symbiotic state, and indicates that at this stage there is more phosphate than mobile PolyP in the AM fungus. The resonance at 30.73 ppm is the hexamethylphosphoramide reference. (From Shachar-Hill et al., 1995).
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In vivo31P spectra of excised roots colonized by G. etunicatum revealed the presence of a polyP peak (at −22 ppm, Fig. 3), whereas spectra of uncolonized roots did not show this peak (Shachar-Hill et al., 1995; Rasmussen et al., 2000). The absence (Shachar-Hill et al., 1995) or presence of very weak signals (Rasmussen et al., 2000) of polyP from terminal and penultimate residues suggests that polyP chains are much longer (> 50 residues) in symbiotic G. etunicatum than in the asymbiotic fungus (see above). Vacuolar and cytoplasmic pH’s (measured from the position of the 31P peaks of Pi in these compartments) as well as nucleotide levels in colonized and control roots were indistinguishable. The mobile polyP content of the colonized root was estimated as equivalent to approx. 10 mM phosphate in the AM fungus, based on a comparison with nucleotide peaks and the assumption that the mycobiont occupies one-tenth of the volume of the host cytoplasm. Despite the small quantities of AM fungal tissue within the host root, the relatively high concentration and unique peaks of fungal polyP mean that NMR spectroscopy can be used to nondestructively detect the presence of AM fungi and can yield information on the nature of the stored fungal polyP (Shachar-Hill et al., 1995; Rasmussen et al., 2000).
Figure 3. The application of NMR to studying arbuscular mycorrhizal P metabolism. In vivo31P NMR spectra of excised one year old leek roots that were either A uncolonized or B colonized with Glomus etunicatum. Shift assignments: a, phosphomonoesters; b, cytoplasmic Pi; c, vacuolar Pi; d, γ phosphate of NTP; e, β phosphate of NTP; f, UDPC; g, α phosphate of NTP; h, central phosphates of polyphosphate. The fungus here occupies only a small fraction of the sample volume and yet its chemical presence is clearly observable (From Shachar-Hill et al., 1995).
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