Aluminium polyphosphate complexes in the mycorrhizal basidiomycete Laccaria bicolor: A 27Al-nuclear...

Planta (1994) 194:241-246 P I ~ L I I t ~

�9 Springer-Verlag 1994

Aluminium polyphosphate complexes in the mycorrhizal basidiomycete Laccaria bicolor: A 27Al-nuclear magnetic resonance study Francis Martin 1, Patrice Rubini 2, Richard C6te 1, Ingrid Kottke 3

Equipe de Microbiologie Foresti~re, Institut National de la Recherche Agronomique, Centre de Recherches de Nancy, F-54280 Champenoux, France 2 Laboratoire de Chimie Physique Organique (LESOC, UA CNRS 406), Facult6 des Sciences, Universit6 de Nancy I, BP 239, F-54506 Vandoeuvre-16s-Nancy Cedex, France 3 Universit/it Tiibingen, Institut ffir Botanik, Spezielle Botanik, Mykologie, Auf der Morgenstelle 1, D-72076 Tiibingen, Germany

Received: 27 September 1993 /Accepted: 31 December 1993

Abstract. The techniques of 27A1- and 31p-nuclear magnetic resonance (NMR) spectroscopy were used to investigate the interactions of aluminium with intracellular ligands within the mycelium of the ectomycorrhizal basidiomycete L a c c a r i a b ico lor (Maire) Orton ($238). The vegetative mycelium was grown on medium containing 0.5 mM A1C13 for 0.5 to 3 d. The 27A1-NMR spectra showed that aluminium was rapidly taken up and accumulated into polyphosphate complexes in the vacuole. Comparison with Al-polyphosphate complexes obtained in vitro on model systems indicated that A1 forms at least three mixed-solvation complexes with Pi and polyphosphates, that there is more than one complex present under any set of conditions, and that the equilibrium between these complexes shifts dramatically with AI concentration in the medium. The high phosphate concentrations in the growth medium favoured the accumulation of the Al-polyphosphate complexes. When mycelium containing Al-polyphosphate complexes was transferred to Al-free nutrient solution for 9 d, the A1- polyphosphate complexes were not remobilized. The sequestration of A1 in the polyphosphate complexes could therefore make a significant contribution to the protection of mycorrhizal plants against aluminium toxicity.

Key words: Aluminium Ectomycorrhizal fungus - L a c -

car la - Phosphate - Polyphosphate

Introduction

Aluminium is one of the most abundant elements in soils and it is occurring in increasing concentrations in the soil solution in acidic soils of Western European forests. Con- cern that A1 toxicity might be a factor in forest decline in Western Europe and North America (Godbold et al.

Abbreviations: NMR = nuclear magnetic resonance; PolyP = polyphosphate(s); PP1 = terminal phosphate of PolyP; PP3 = middle phosphate of PolyP Correspondence to: F. Martin; FAX: (33) 83394069

1988) has led a number of workers to investigate A1 metabolism in trees. Toxicity to plants and microbes of one or more of the chemical species of A1 has been known for many decades. However, little is known concerning the physiological mechanisms responsible for stress tolerance or susceptibility in soil microbes and plants (Wool- house 1983; Foy 1984). Despite the complex nature of A1 toxicity in plants, considerable evidence indicates that it is primarily associated with a disruption of root physiology and function. The initial symptom of A1 toxicity is an inhibition of root growth with subsequents effects on nutrient and water uptake (Haug 1984; Kochian and Shaft 1991). Recent studies (Huang et al. 1993) have indicated that continuous A1 disruption of Ca 2+ absorption into the root apex cells of Al-sensitive wheat cultivars could alter Ca 2+ nutrition and homeostasis in these cells and play a pivotal role in the mechanisms of A1 toxicity. In several instances, roots have been shown to reduce A1 toxicity by binding A1 to root surfaces. This metal-ion trapping (biosorption) is a well-known feature of the cell walls of microbes and plants (Horst et al. 1983; V/ire 1990; Guida et al. 1991; Hodson and Wilkins 1991). Ex- cretion of organic acids (e.g. malic acid) that chelate and detoxify AI external to the symplasm has also been iden- tified as a possible mechanism of A1 tolerance (Delhaize et al. 1993a,b).

The presence of ectomycorrhizal fungi on tree roots modifies the compartmentat ion of the absorbed A1 and protects the host plant against the toxic effects of A1 (Cumming and Weinstein 1990; V/ire 1990; Hodson and Wilkins 1991; Wilkins 1991), albeit this opinion has been recently challenged (Jentschke et al. 1991). To explain this finding, it was proposed that polyphosphates (PolyP), lin- ear condensed polymers of inorganic phosphate, se- quester heavy-metal ions and A1 as part of the fungal detoxification mechanism (V/ire 1990; Kottke 1991). Polyphosphates are the only macromolecular anions in the fungal vacuole (Cramer and Davis 1984; Klionsky et al. 1990), and their roles in the retention of basic amino acids and cations and in osmoregulation have been demonstrated both in vivo and in vitro (Miller~1984;

242 F. Martin et al.: Aluminium-polyphosphate complexes in Laccaria

K l i o n s k y et al. 1990). In several species of cyanobac te r i a , unice l lu lar euka ryo t i c algae, and ec tomycor rh i za l fungi an a s soc ia t ion of heavy-me ta l ions (cadmium, u ran ium) and A1 with p h o s p h o r u s (P)-rich vacuo la r bodies has been d e m o n s t r a t e d using energy-d ispers ive X - r a y ana ly - sis (Pe t te rsson et al. 1985; Vfire 1990) and e lec t ron-energy- loss imag ing (Kot tke 1991).

In aqueous solut ions , A13+ forms an oc t ahedra l hex- ahydra te , AI(H20)63+ at acid pH. W h a t l igands might b ind A13+ in the fungal cell? The mos t l ikely A13+-bind - ing sites are oxygen a toms, especia l ly if they are negat ively charged. C a r b o x y l a t e and p h o s p h a t e g roups are the s t ronges t A13+ binders , and A13+ will f requent ly form soluble complexes with p h o s p h a t e g roups in b io logica l sys tems (Woolhouse 1983; M a r t i n 1992). In the myce l ium of e c t o m y c o r r h i z a l fungi, P o l y p m a y serve as a poo l of reserve phospha te , ava i lab le for hydrolys is , with subsequent t ransfer to the hos t p l an t as Pi fol lowing d e m a n d by the hos t p lan t (Mar t in et al. 1983, 1985; M a c F a l l et al. 1992). This m o d e l w o u l d pred ic t tha t under cond i t ions in which P is l imi ted to the p lan t (e.g. forest soils), there would be litt le or no Po lyP a c c u m u l a t i o n by the fungus. However , M a c F a l l et al. (1992) d e m o n s t r a t e d tha t even under soil cond i t ions which are P- l imi ted for seedling growth , s ignif icant P is a c c u m u l a t e d as Po lyP by the fungal symbion t . P o l y p h o s p h a t e m a y well become an impor - tan t A13 + binder . The use of 27A1-NMR spec t roscopy is the only definit ive means of ident i fying A1 species in solu- t ion (Kar l ik et al. 1982, 1983). However , re la t ively few studies r epor t the use of 27A1-NMR in aqueous so lu t ion with l igands of b io log ica l in teres t (Kar l ik et al. 1982, 1983; N a g a t a et al. 1991, 1993). Since the ec tomycor - rhizal fungus L. bicolor con ta ins a high level of Po lyP (Mar t in 1991) and can a d a p t to g row in a m e d i u m con- ta in ing high a concen t r a t i on of A1CI 3 (D.A. Wilkins , School of Biol. Sciences, Un ive r s i ty of B i rmingham, U K , pe r sona l communica t ion ) , we invest igated, by 31p_ and 27AI-NMR, if the fo rma t ion of A1-PolyP complexes oc- curs in its vegeta t ive mycel ium.

Materials and methods

culture as a function of time, rapidly harvested by filtration, and analyzed by NMR (Martin 1991). For each experiment, approx. 0.7-1.0 g mycelium was required; this was generally examined by NMR within 5-10 min of filtration. Experiments were carried out in triplicate on separate occasions.

Aluminium solutions. The products obtained by mixing A1 salts with various ligands are dependent on the order of mixing. In the present study, all compounds were added to the A1 solution at pH 5.5 (the pH of the growth medium and the fungal vacuole). The total A1 concentration of the medium and fungal extracts was monitored by an inductively coupled plasma spectrometer (ICP, Jobin Yvon, Paris, France) on perchloric digests. Aluminium speciation was cal- culated using the computer software GEOCHEM-PC (Parker et al. 1991). According to these estimates, free AI 3+ was the prominent species (51%) and the remaining A1 was complexed with sulphate (23%), phosphate (14%), tartrate (7%) and hydroxyl (5%) ions. Solutions of AI (poly)phosphate were prepared by mixing 5 mM aqueous solutions of Polyp (n = 3, 5, or 15) (Sigma Chimie, St Quentin, France) and 5 mM A1C13.

Nuclear magnetic resonance spectroscopy. High-resolution 3Jp. and 27A1-NMR experiments were performed on a 9.4T Bruker AM-400 spectrometer (Nuclear Magnetic Resonance Unit, University of Nancy I, Vand0euvre-l~s-Nancy, France) operating at 161.9 and 104.2 MHz, respectively, under nonsaturating conditions. The 31P-NMR spectra of intact mycelium and aqueous solutions were obtained by using a pulse width (45 ~ of 17 ~ts, 10 kHz spectral width, 16 Kbytes data storage array, and a recycle time of 0.82 s. The 27AI-NMR spectra of intact mycelium and aqueous solutions were obtained by using a pulse width (90 ~ of 17 gs, 8 kHz spectral width, 8 K data storage array, and a recycle time of 64 ms (1 K acquisition data). The free induction decays (20000 pulses) were exponentially multiplied with a line broadening of 5-10 Hz prior to Fourier transformation. External 100mM A1C13 and 85% orthophosphoric acid were used as standards, and chemical shifts were referenced relative to these standards which were assigned a value of 0 ppm. Complexed AI observable by NMR was quantitat- ed by comparison of the integrated peaks from the mycelium spectra with the integrated peaks from a 5-mM solution of A1C13-PolyP (n = 15). The average standard deviation for three replicate experiments was not greater than 15%.

Estimation of the intracellular pH. Measurements of intracellular pH from cytoplasmic and vacuolar Pi, and Polyp terminal P were made using the standard reference curve of pH versus 31p chemical shift in ppm (Roberts et al. 1981). Solutions prepared for evaluation of this pHprofile contained 10mM K2HPO4, 10mM PolyP (n = 15), 2 mM MgC12 and 100 mM KC1.

Mycelium growth. Culture of Laccaria bicolor (Maire) Orton (isolate $238; = L. laccata $238) was provided by Dr. R. Molina (U.S. Forest Service, Corvallis, Oreg., USA) and maintained in the collec- tion of ectomycorrhizal fungi at the Forest Microbiology Unit (I.N.R.A.-Nancy, Champenoux, France). To evaluate the metabolism of A1 in the mycelium, it was essential that we investigate these effects at a pH between 4 and 5 since A1 becomes mobile in the soil in this pH range. Furthermore, this pH range favours the formation of the toxic species, A13 § (Martin 1992). The mycelium was cultivated in liquid cultures on Pachlewski's medium (1.0 mM KHaPO4, 100 mM glucose, 2.7 mM di-ammonium tartrate, 7.3 mM MgSO 4, 7H20, 2.9 gM thiamine-HCl and 0.1% (v/v) of a trace-element stock solution (Kanieltra 6Fe, Hydro Azote Specialit~s, Cergy Pontoise, France) as described previously (Martin et al. 1983; Mar- tin et al. 1985). Some investigations were carried out with Pach- lewski's medium containing 0 or 7.3 mM KHzPO 4. When the culture reached the middle of the rapid phase of growth (after approx. 10 d), it was collected by filtration and transferred to a fresh Pach- lewski's medium containing 0.5 mM AICI 3 for 0.5 3 d. As a result of ammonium uptake, the pH of the medium rapidly reaches a value of 4. Aliquots of mycelium were removed subsequently from the

Data processing. Deconvolution of the spectra of mixed solvation complexes of A1-PolyP into the various constitutive components was performed by simulation of the experimental spectra considered as the sum of Lorentzian functions. The population, linewidth and frequency of each NMR site were adjusted by an in-house computer software in order to obtain the best fit with the actual spectrum.

Results

Polyphosphate content and localization. The myce l ium of L. bicolor accumula t ed a large a m o u n t of p h o s p h a t e in the form of Po lyp when g rown on 1 m M Pi (Fig. 1). The resonance ass ignments for these spec t ra are as p rev ious ly r epor t ed (Mar t in et al. 1983, 1985; Mar t i n 1991). The te rmina l (PP1), penu l t ima te (PP2), and midd le (PP3) phospha tes of Po lyp resona te at - 6 . 2 , - 2 0 . 0 , and - 2 2 . 3 ppm, respectively. The average chain length of

F. Martin et al.: Aluminium-polyphosphate complexes in Laccaria 243

e-

ffl e- O3

If,0-

1 0 0 -

Pic~ P~vac A

0-

PF

.2'o ppm

Fig. 1. In-situ 3'p-NMR spectra of Laccaria bicolor grown on 0, 1 and 7 mM Pi medium. The mycelium was grown on the different Pachlewski's media for 5 d. Each spectrum represents 1000 scans accumulated over approx. 30 min and processed using line broadening of 10 Hz. Pi~y~, cytoplasmic inorganic phosphate; Pi ...... vacuolar inorganic phosphate; PP~, PP2, and PP3, terminal, penultimate, and middle P in PolyP, respectively. Chemical shifts are expressed in ppm from 85% orthophosphoric acid at 0 ppm

these PolyP was approx 25-30 P residues, as determined from the PPJPP1 ratio (Martin et al. 1983). They contained up to 80% of the NMR-observable P in the mycelium and were stored in the cell in a soluble form, but with a restricted molecular freedom, as suggested by the T~ relaxation time of the middle P resonance (70 ms; see Martin et al. 1985). When the mycelium was subcul- tured on a P-deficient medium for 5 d, the concentration of the intracellular PolyP decreased markedly (Fig. 1), but PolyP were still detectable. In contrast, when it was cultivated under P-rich conditions (7 mM P), PolyP accumulation increased, supporting the idea that these polymers have a storage function.

The chemical shifts of P i ~ and Picy t predict a pH of 5.6 and 6.8 for the vacuole and the cytosol, respectively, as estimated from the in-vitro pH-Pi chemical shift titration curve (data not shown). The chemical shift of PP1 is also pH dependent (Navon et al. 1979; Shanks and Bailey 1990; Beauvoit et al. 1991). The chemical shifts of PP1 predict a pH of 6.0, as estimated from the pH titration curve for PP~ (data not shown). This acidic pH value supports a vacuolar compartmentat ion of the NMR-observable PolyP.

27A1-Nuclear magnetic resonance spectra of Al-polyphosphate complexes. As a first step, we have probed the interaction of AI 3+ with phosphate and PolyP in aqueous solutions using 27AI-NMR. Although the 2VA1 nucleus is quadrupolar (spin 5/2), the combination of high natural abundance (100%) and sensitivity (0.2 relative to proton) readily allows observation of complexes in the millimolar concentration range (Fig. 2). The complexes of these ligands with A13+ occurred in slow exchange with Al(H20)63+ on the 27A1-NMR time scale, and can be observed as peaks that are distinct from the resonance of Al(H20)63+ (peak i), allowing the determination of the number of complexes. Binding of AI 3+ to PolyP produced upfield shifts of 3-7 ppm from Al(H20)63+ in the 27A1 spectrum as illustrated in Fig. 2B,C. The spectrum observed for A1 and Pi (Fig. 2A) consists of a sharp reso-

2 3 ~ . _ _ _ A 4

5

4 / t

/ / "\

1'0 b -iO -;20 ppm Fig. 2. 27A1-Nuclear magnetic resonance spectra for equimolar mix- tures of 5 mM A1CI 3 and 5 mM orthophosphate (A), 5 mM PolyP with an average chain length of 5 (B), and 5 mM PolyP with an average chain length of 15 (C) at pH 5.5 and 20~ recorded at 101.4 MHz (number of scans = 1000). Chemical shifts expressed in ppm from AI(H20)63+ at 0 ppm

nance (Av ~ 7 Hz) at 0 ppm (peak 1), corresponding to Al(H20)63+, and two overlapping broad resonances observed upfield at - 1.2 (peak 2) and - 3.2 ppm (peak 3) with linewidths of 150 and 260 Hz, respectively. Observed shifts are indicative of direct binding of A1 to phosphate (Karlik et al. 1983). The more downfield resonance is due to a 1 : 1 complex of A1 and phosphate, whereas the more upfield resonance is presumably due to a complex with a P/AI ratio > 1 (Karlik et al. 1982). When A1 was mixed with a short-chain-length PolyP (n = 5) in equal propor- tions, the Al(H20)63+ species was not detectable (Fig. 2B). All of the 27A1 was observed as a broad peak composed of two overlapping resonances, one relatively sharp (Av ~ 50 Hz) at --3.4 ppm (peak 4) and a large hump (Av ~ 500 Hz; peak 5). Aluminium complexes with tripolyphosphate only generated the resonance at - 3 . 4 ppm (data not shown). The upfield shift (peak 5) was consistent with the A1-NMR shifts observed for mixed-solvation A13+ complexes with orthophosphate (Karlik et al. 1982), trimethyl phosphate (Delpuech 1983), and a variety of phosphate-containing ligands (Karlik et al. 1983). The affinity of the PolyP for A1 increased with the number of P residues present in the PolyP chain as shown by the decrease of the "free" Al(H20)63+ resonance (Fig. 2B,C). As the chain length of the PolyP increased (n -- 15), the initially sharp resonance (peak 4) broadened and the 27A1 signal was observed as two overlapping broad resonances at - 3 . 4 and - 7 . 3 ppm (Fig. 2C). An increase in the number of available phosphate groups for coordination produced an increase in


the amount of complexed A1. The broadening of the resonance of bound species presumably results from a decrease in the symmetry of the complex as a result of the substitution of P for H20 in the A13 + coordination sphere (Karlik et al. 1982), inducing a higher quadrupolar relaxation rate of the 27A1 nucleus. Detailed N M R studies of A13 +-PolyP complexes formed in vitro will be described elsewhere.

Mixed-solvat ion complexes o f Al in Laccaria. When L. bicolor mycelium was supplied with a medium containing 0.5 mM A1C13, A1 uptake and formation of A13+-phos - phate complexes were rapidly initiated. Representative 27A1 spectra recorded in situ during this process are shown in Fig. 3. Interestingly, the A1 signals increased only at the positions typical of the A13 +-PolyP complexes (see above). The 27A1 signals were observed as two overlapping broad resonances at - 3 . 4 (peak 1) and - 7 . 3 ppm (peak 2). The resonance shape and linewidth (of approx. 50 and 500 Hz) indicated that the signals orig- inated from complexes of A1 and PolyP with a chain length > 5 phosphate residues (compare Fig. 3C and Fig. 2B). No signal corresponding to Al(H20)63+ (at 0 ppm), organic acid (tartrate, malate, citrate; 0 to + 50 ppm) or AI(OH) 4 (+ 80 ppm) complexes was observed. The formation of the A1-PolyP complexes was rapid; that is, within the first hours following the addition of A1C13 (Fig. 3B). Increasing the incubation time showed that the component at - 3 . 4 ppm (peak 1) remained at the same concentration, while the wider upfield components (peak 2) continued to increase (Fig. 3C,D). Thus, with increased A13 + availability the transition to polymeric species was favoured. Much larger accumulations of A13+-PolyP complexes were obtained when the mycelium was grown on 5 mM A1C13 for 3 d (Fig. 4). Computer analysis of the A13+-PolyP resonances was performed to extract addi- tional information about the types of complex accumu-

1

1

2 2

J ~ ,c

6 -S ppm

Fig. 3. In-situ 27A1-NMR spectra of mixed-solvation A1 complexes in L. bicolor. The mycelium was grown on a medium containing 1 mM Pi, together with 0.5 mM AIC13 for the specified time. A, Control, no A1C13; B, 0.5 d; C, 1 d; and D, 3 d of Al-influence. Each spectrum represents 20000 scans accumulated over approx. 30 min and processed using line broadening of 10 Hz. The arrow (~,) indicates the chemical shift of AI(HaO)63+ at 0 ppm

%2

I -5 -10 ppm

Fig. 4. Deconvolution analysis of the Al-polyphosphate resonance observed in L. bicolor grown on 5 mM A1C13 for 3 d. The peak could be fitted using four overlapping Lorentzian curves. AI(PO4)n(H20)6 n refers to A1 interacting with one to three P residues of the same PolyP chain or stacked PolyP chains

lated. The composite peak could be fitted using four overlapping Lorentzian curves (Fig. 4). The less abundant component (4%) represents unbound Al(H20)63+. The other components originate from A13+-PolyP complexes with increasing P/A1 ratios. Peak 1 (15%) may correspond to A1 interacting with the terminal P residue of the Polyp chain, whereas peak 2 (49%) and peak 3 (32%) may correspond to A1 interacting with two P residues of the same Polyp chain or stacked structures where A1 acts as a bridge between different Polyp chains (Karlik et al. 1982). A comparison of the N M R spectra of mixed-solvation A13 +-PolyP complexes observed in vitro with those obtained in intact mycelium indicated that A13 + forms at least three complexes with Pi and PolyP, that there is more than one complex present under any set of conditions, and that the equilibria between these complexes shift dramatically with A1 concentration in the medium.

The calculation of complex concentration was made from the area of the corresponding resonances with reference to A1-PolyP (n = 15) observed in vitro, the concentration of which is known. Accumulation of the mixed- solvation complexes increased with P and A1 concentration in the growth medium. In mycelium grown on 1 mM P and 0.5 mM A1C13 for ! d, the A1 concentration in the mycelium was 9.2 mM, whereas A1 sequestered in the mixed-solvation Polyp complexes within the mycelium reached 28 mM.

The turnover of the A1-PolyP complexes was tested. Mycelium grown on 0.5 mM A1C13 for 3 d and thus containing A1-PolyP complexes was transferred to Al-free nutrient solution for 9 d. The concentration of NMR-observable A1-PolyP complexes remained constant (Fig. 5), indicating that AI sequestrated in the complexes was not readily remobilized. In mycelium grown on P-depleted growth medium the PolyP content decreased as shown

F. Martin et al.: Aluminium-polyphosphate complexes in Laccaria 245

0 -S ppm

Fig. 5. Stability of the A1-PolyP complexes. The mycelium of L. bicolor was grown on l m M P and 0.5 mM A1CI 3 for 3 d and then transferred to Al-free nutrient solution for specified time. A, 0 d; B, 3d; C, 9 d

above (Fig. 1) and accumulation of A1-PolyP complexes was not observed (data not shown).

Discussion

Before we can understand the effect of A1 on cellular metabolism we need to learn how it interacts with molecules found in biological systems (Martin 1992). The aim of the present study was to determine the intracellular ligand of AI in the ectomycorrhizal basidiomycete Laccaria bicolor. Cellular compartmentation of heavy metals and A1 in ectomycorrhizal fungi has been investi- gated by energy-dispersive X-ray microanalysis and electron-energy-loss imaging (Denny and Wilkins 1987; V/ire 1990; Kottke 1991). Aluminium was detected in P-rich granules within the vacuoles (Kottke 1991) and cells walls (V/ire 1990) and it has been suggested that exclusion at the cell wall is the primary mechanism for protection of fungal hyphae (V/ire 1990).

The 27A1-NMR data presented here have shown for the first time that mixed-solvation complexes of A1-PolyP can occur in the vacuole of the fungal cells. Other cellular ligands (e.g. organic acids) may occur in the fungal cell, but A1 absorbed by rapidly growing L. bicolor is efficient- ly and prominently sequestered in the Polyp complexes, indicating that the Polyp sequestration is the main con- tributor to the A1 tolerance of the fungal strain in our study. The current data indicate that the Al-PolyP complexes observed by N M R correspond to the P-rich granules containing A1 detected by microscopy (Kottke 1991). The comparison of spin-lattice relaxation time, T~, of Polyp in situ (70 ms) with those observed in model solutions (Martin et al. 1985) confirmed that PolyP occurred in L. bicolor in a soluble form with reduced correlation time. This restricted molecular freedom is likely due to interchain interactions and interactions with cellular ions, such as A13+.

For the following reasons, we propose that A1 sequestration by vacuolar Polyp protects the fungal cell and associated roots from A1 toxicity: (i) PolyP interact with absorbed A1 within a few hours; (ii) A1-PolyP complexes accumule in the mycelium grown on a wide range of A1 concentrations (0.5 5 mM); (iii) accumulated A1-PolyP complexes are not dissociated when the mycelium is

transferred to Al-free medium, suggesting that A1 is effi- ciently immobilized; (iv) the ectomycorrhizal mantle en- sheathing short roots is a primary site of PolyP accumulation (Kottke 1991; Wilkins 1991); and (v) the presence of ectomycorrhizal fungi on roots modifies the compartmentation of the absorbed A1 and protects the host plant against the toxic effects of A1 (Cumming and Weinstein 1990; V/ire 1990; Hodson and Wilkins 1991; Wilkins 1991).

In freeqiving mycorrhizal fungi grown on P-sufficient medium and in ectomycorrhizal roots, PolyP are the major P-storage compounds and are available for catabolism with subsequent transfer to the host plant as Pi following demand by the host plant (Martin et al. 1983, 1985; MacFall et al. 1992). The vacuolar accumulation of soluble A1-PolyP complexes in the mycelium is likely to occur in the conditions where significant P concentrations together with toxic A13+ concentrations occur in soils. Forest soils are characterized by a low Pi concentration in solution (1 10 ~tM) and it appears unlikely that the extramatrical mycelium permeating the soil could ac- cumulate Al-PolyP complexes at this concentration. In contrast to this contention, significant amounts of PolyP are present in the ectomycorrhizal mycelium grown on 5 pM Pi (Lapeyrie et al. 1984) and in mycorrhizal roots of Pinus resinosa grown under P-limiting conditions (Mc- Fall et al. 1992). This supports a role for PolyP in binding A1 at the point of uptake. The extramatrical mycelium is certainly a major site of metal binding, rather than the mantle and Hartig net (Denny and Wilkins 1987). Alter- natively, the sequestration of A1 at the interface between the fungus and the host plants presumably plays a part in the detoxification mechanism. Under natural conditions, where the effect of the transpiration stream will be to draw Pi, PolyP and A1 along the extramatrical hyphae towards the roots (for a discussion see Shepperd et al. 1993), the ectomycorrhizal short roots may represent the final barrier to the movement of A1 toward the plant. The mantle and the interface region are particularly rich in PolyP and are presumably major sites of A1-PolyP complexes in ectomycorrhizal associations.

We provide evidence to support the hypothesis that AI-PolyP accumulation is one of the possible Al-tolerance mechanisms used by fungal cells and ectomycorrhizal plants. However, the AI trapped in Polyp may af- fect the generation of phosphate, disturb the translocation of other cations (e.g. arginine, K § by preventing their binding to PolyP and subsequent transport. The role of A1 sequestration in fungal metabolism must therefore be carefully considered. Investigations are currently being carried out on the turnover rate of these complexes in relation to the nutrient levels, the competition with other cellular ligands, and the transfert of A1 to the cell roots when the Polyp are degraded at the symbiotic interface.

Aluminium metabolism in fungal and plant cells is of considerable importance, and in-vivo NMR of intact or perfused cells provides an excellent means for studying it, particularly in view of the fact that no alternative ap- proach allows the quantitation of the various A1 species in living cells. The information gained on Polyp and A1 interactions in L. bicolor demonstrates the potential of this technique for future investigations of A1 metabolism.


We thank Prof. Daniel Canet (Laboratoire de M6thodologie RMN, University of Nancy I, Vand~euvre-16s-Nancy, France) for his constant encouragement and Christine Delaruelle for skilled technical assistance in growing the fungal cultures. This work was supported by a research grant from the Commission of the European Commu- nities (STEP-CT90-0059, "Role of Ectomycorrhiza in Stress Toler- ance of Forest Trees") to F.M. and a travel grant from the Institut National de la Recherche Agronomique to I.K.; R.C. is a recipient of a Postdoctoral Fellowship from the Natural Sciences and Engi- neering Research Council of Canada.

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