ULTRA-STRUCTURE AND MICRO ANALYSIS OF THE POLYPHOSPHATE GRANULES OF THE ECTOMYCORRHIZAS OF FAGUS SYLVATICA

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1 New Phytol. (1982) 92, ULTRA-STRUCTURE AND MICRO ANALYSIS OF THE POLYPHOSPHATE GRANULES OF THE ECTOMYCORRHIZAS OF FAGUS SYLVATICA BY D. G. STRULLU*, J. L. HARLEYf, J. P. GOURRETJ AND J. P. GARREC Laboratoire de Biologie Vegetale, Universite d'angers, Angers Cedex, France,* Department of Agricultural and Forest Sciences, University of Oxford, Parks Road, Oxford OXl PF, U.K.,i Laboratoire de Biologie Cellulaire, L.A. n 256 C.N.R.S., Universite de Rennes, 5042 Rennes Cedex, FranceX and Laboratoire de Biologie Vegetale, C.E.N.G, 8042 Grenoble Cedex, France^ {Accepted 11 June 1982) SUMMARY Evidence is presented that the granules visible in the vacuoles of the hyphae of the sheath of Fagus sylvatica mycorrhiza in electron microscope (EM) preparations contain phosphorus and calcium. Little phophorus or calcium is present in the clear regions of the hyphal vacuoles. Calcium but not phosphorus is present in considerable quantity in the hyphal walls of the fungal sheath. INTRODUCTION Granules of polyphosphate have now been observed in many types of mycorrhiza. These structures were first observed in the mycorrhizas of Pinus brutia where they were called metachromatic granules by StruUu and Gourret (197). Ashford, Ling Lee and Chilvers (1975) made a detailed cytochemical study and called them polyphosphate granules. Chilvers and Harley (1980) showed that the incubation of mycorrhizal roots of beech in potassium phosphate solutions resulted in the increase o"^ bc-l-k +ke f^<sv>rshe.r a<\ti 4^^ s\ze <^ gr^c^ves <xs vvev-o^d uj'<-\vv -VW.. U^ microscope. Harley and McCready (1981) demonstrated that over an absorption period of 19 h the proportion of absorbed ^^phosphate converted into polyphosphate increases with external phosphate concentration. StruUu, Gouret and Garrec (1981a) made a microanalytical study of the polyphosphate granules in the mycorrhizas of Pseudotsuga menziesii and found them to be rich in both phosphate and calcium. In addition, similar types of polyphosphate granules have been described in various endomycorrhizas where they have been subjected to microanalysis by White and Brown (1979), Cox et al. (1980) and Strullu et al. (1981b). The purpose of this paper is to examine the granules in the fungal sheath of the mycorrhizas of Fagus sylvatica and to learn something of their content of phosphorus and other ions. MATERIALS AND METHODS Samples of the mycorrhizas of Fagus were collected from the litter layer of the beech forest near Christmas Common in the Chiltern Hills, England in late X/82/ $0.00/ The New Phytologist

2 4i8 D. G. STRULLU et al. September. The apical regions were excised and washed to clear them of soil particles. The fungal sheath of some was immediately separated from the host tissue by dissection under a binocular microscope. Small pieces of mycorrhiza cut lengthwise together with the sheath tissue obtained by dissection were fixed in 2-5% glutaraldehyde buffered at ph 7-2 with 01 M cacodylate for 5 h to ensure that the fixative penetrated the close-textured fungal tissue. The tissues were post-fixed for 1 h in 2 % osmium tetroxide in the same buffer. After dehydration the material was embedded in epoxy resin. Sections between 1000 and 5000 A thick were cut, but those of 1800 A were used for studying structure. The fixation appeared to be quite satisfactory. For the electron probe microanalysis, sections of different thicknesses were deposited on copper or aluminium grids with a collodion- and carbon-coated support film. They were studied with a Camebax electron probe (Strullu et al. 1981a, b). The instrument operated at 45 kv accelerating voltage, 120 na specimen current and a probe diameter of 00 nm. RESULTS Ultrastructure of the mycorrhizas and presence of granules The mycorrhizas possessed a sheath consisting of an external plectenchymatous layer of compacted hyphae. Within it, the inner plectenchymatous layer consisted of more loosely separated hyphae. The inner layer was particularly studied, for in it the cells of the separate hyphae were well organized. The fungus in this mycorrhiza was a basidiomycete, as evidenced by its dolipore septa [Fig. l(c)], and the organization of the walls of its hyphae was similar to that of the basidiomycetous mycorrhiza of Pseudotsuga (Strullu, 1979). The hyphae possessed a protoplast which appeared to be living. The plasmalemma was wrinkled; the cytoplasmic membrane systems and the organelles were well developed and preserved. The clear vacuoles were bound by a characteristic tonoplast and contained one or more granules which were electron-dense and similar to polyphosphate granules of other material. These granules volatilized under the electron beam during microscopic observation. An examination of a range of sections of different thicknesses between 1000 and 5000 A shows that the rate of disappearance of the granules depends on this parameter. The volatilization is very rapid from sections of 1000 A thickness, but the initial location of the granule remains visible in the vacuole (Fig. l(a)]. In sections of 5000 A thickness the vacuoles can be seen to contain 1-10 granules which, with few exceptions, do not volatilize even after a long period of observation [Fig. 2(b)]. Microanalysis The spectrum of energy dispersion is shown in [Fig. l(d)]. The peaks correspond to phosphorus, sulphur, chlorine, potassium and calcium. Other Fig. 1. (a) Electron microscopic - glutaraldehyde-osmium fixation (x55oo). Fungal mantle of Fagus mycorrhizas showing loosely separated hyphae. The granules present in vacuoles (v) volatilize very rapidly from this section of 1000 A thickness. The initial location of the granule remains visible as a hole in the vacuole (large arrows). The presence of glycogen particles (g) can also be noted, (b) In section of 5000 A thick the vacuoles contain 1-10 granules which, generally, do not volatilize (large arrows). (x8000). (c) Electron microscopic - glutaraldehyde-osmium fixation (x ). Typical dolipore-parenthesome septum of basidiomycetous fungi. (d) Spectrum of energy dispersion of polyphosphate granule. Peaks correspond to phosphorus (P), sulphur (S), chlorine (Cl), potassium (K) and calcium (Ca).

3 Polyphosphate granules in Fagus J ' J^ =^* \:.^ hi Fig

4 42O D. G. STRULLU et al. Fig. 2. (a) Secondary image of a 1800 A thick section ( x 8000). Notice the presence of two granules of different sizes (arrows) present in vacuole (v), (fw), wall of the fungal hyphae. (b) Phosphorus-X image corresponding to (a). Two white spots (arrows), corresponding to the granules, signal the presence of phosphorus. elements might equally have been detected but their presence may be dependent upon conditions. Microanalysis by wavelength dispersion was used to compare relative concentrations of phosphorus and calcium in different parts of the hyphae. The results are given in Table 1. The following conclusions may be drawn. The granules of

5 Polyphosphate granules in Fagus 421 Table 1. Relative concentrations of phosphorus and calcium in selected points of Fagus sylvatica mycorrhizas {counts 10 s~^) Granule Light part of the vacuole Fungus-wall P Ca P Ca P Ca (0) (9) ( ) = Concentration noted in interhyphal zone. polyphosphate in the mycorrhizas of beach contain both phosphorus and calcium. Both these elements are present at far lower concentrations in the clear zones of the vacuole. The walls of the mycorrhizal hyphae are rich in calcium, which is present in only very minor quantities in the interhyphal zones. The secondary electron images shown in Figure 2(a) indicate two polyphosphate granules of different sizes (about 04 and 0-8 /*m, arrowed), each one present in a vacuole. The walls of the fungal hyphae can be recognized (FW). The phosphorus-x images of these features are shown in Figure 2(b), where the group of white spots (arrows) indicates that phosphorus is abundant in the granules. Figures (a) and (b) superimpose the line of analysis of the section and the phosphorus or calcium content on the secondary electron image of the fungal cells. From Figure (a) it is clear that the phosphorus content of the granules is high, as shown by the two peaks, each over a granule. Phosphorus is present in small quantity only, in the walls of the hyphae (asterisk) but occurs in variable quantity in the cytoplasm (circle). Calcium [Fig. (b)] is also present in high quantity in the granules as shown by its peaks. The figure also shows peaks of occurence of calcium in the hyphal walls (asterisks) but relatively low amounts in the cytoplasm (circle). DISCUSSION As pointed out by Strullu et al. (1981a, b), vacuolar granules containing phosphate and calcium occur in a number of symbiotic systems, for instance in ectomycorrhizal Pseudotsuga and endomycorrhizal Taxus, Trifolium and Pellia. The methods of microanalysis using wavelength dispersion are sufficiently sensitive for the study of structures of the order 0-5 /*m. The chemical elements in them may be studied singly, or in pairs by using two spectrometers simultaneously. In addition, by using a diode it is also possible to obtain, with the same type of apparatus, the energy dispersion spectrum of a number of elements using fixed samples. The glutaraldehyde and osmium used here gives good preservation both of the mycorrhizal partners and of the polyphosphate granules. It may be that there is some change in the distribution of the elements during fixation (Strullu et al. 1981a). This is being examined using methods which allow examination of the granules at high resolution without preliminary fixation. The

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7 Polyphosphate granules in Fagus 42 present observations, however, show that in Fagus ectomycorrhizas, as in others tested, granules rich in phosphate and in calcium occur in the fungal vacuoles. Vacuoles storing polyphosphate may possibly act in ectomycorrhizas, as in endomycorrhizas (Strullu et al. 1981a), as a cation trap. Following these data regarding the composition of polyphosphate granules additional information should now be sought about the role of cations in the accumulation of polyphosphate. ACKNOWLEDGEMENTS The authors thank Mrs G. Boguais of the Laboratoire de Biologie Cellulaire, M. C. Rousset of the Laboratoire de Botanique (Universite de Rennes) and M. J. P. Bossis (C. E. N. Grenoble) for their technical assistance. REFERENCES ASHFORD, A. E., LING LEE, M. & CHILVERS, G. A. (1975). Polyphosphate in eucalypt mycorrhizas; a cytochemical demonstration. New Phytologist, 74, CHILVERS, G. A. & HARLEY, J. L. (1980). Visualization of phosphate accumulation in beech mycorrhizas. New Phytologist, 84, Cox, G., MoRAN, K. J., SANDERS, F., NOCKOLDS, C. & TINKER, P. B. (1980). Translocation and transfer of nutrient in vesicular-arbuscular mycorrhizas. IIL Polyphosphate granules and phosphorus translocation. New Phytologist, 84, HAKLEY, J. L. & MCCREADY, C. C. (1981). Phosphate accumulation in Fagus mycorrhizas. New Phytologist, 89, STRULLU, D. G. (1979). Ultrastructure et representation spatiale du manteau fongique des ectomycorrhizes. Canadian Journal of Botany, 57, STRULLU, D. G. & GOURRET, J. P. (197). Etudes des mycorrhizes ectotrophes de Pinus brutia. Ten. en microscopie electronique a balayage et a transmission. Compte rendu Hebdomadaire des seances de VAcademie des Sciences, 111, serie D, STRULLU, D. G., GOURRET, J. P. & GARREC, J. P. (1981 a). Microanalyse des vacuolaires des ectomycorrhizes, endomycorrhizes et endomycothalles. Physiologie Vegetale, 19, STRULLU, D. G., GOURRET, J. P., GARREC, J. P. & FOURCY, A. (1981b). Ultrastructure and electron probe microanalysis of the metachromatic vacuolar granules occurring in Taxus mycorrhizas. New Phytologist, 87, WHITE, J. A. & BROWN, M. F. (1979). Ultrastructure and X-ray analysis of phosphorus granules in a vesicular-arbuscular mycorrhizal fungus. Canadian Journal of Botany, 57,

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