AND J. DEXHEIMER. Station d'amelioration des Plantes, INRA, BV 1540, Dijon-CedeXy France

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New Phytol. (1979) 82, 127-132 ENZYMATIC STUDIES ON THE METABOLISM OF VESICULAR-ARBUSCULAR MYCORRHIZA. III.

1 New Phytol. (1979) 82, ENZYMATIC STUDIES ON THE METABOLISM OF VESICULAR-ARBUSCULAR MYCORRHIZA. III. ULTRASTRUCTURAL LOCALIZATION OF ACID AND ALKALINE PHOSPHATASE IN ONION ROOTS INFECTED BY GLOMUS MOSSEAE (NICOL. & GERD.) BY S. GIANINAZZI, V. GIANINAZZI-PEARSON Station d'amelioration des Plantes, INRA, BV 1540, Dijon-CedeXy France AND J. DEXHEIMER Laboratoire de Botanique II, Universite de Nancy, Nancy-Cedex, France {Received 15 March 1978) SUMMARY The ultrastructural distribution of acid and alkaline phosphatase in 6-week-old onion roots infected by Glomus mosseae has been investigated cytochennically. Significant acid phosphatase activity was only observed in the little vacuolated, immature terminal arbuscule branches of the mycorrhizal fungus whilst strong alkaline a-naphthyl phosphatase and /?-glycerophosphatase activities were localized within the vacuoles of the mature arbuscular and intercellular hyphae. In the host cells neither acid nor alkaline phosphatase distribution was modified with vesiculararbuscular mycorrhiza formation. These results are discussed in relation to previously reported mycorrhiza-specific alkaline phosphatase (Gianinazzi-Pearson and Gianinazzi, 1976, 1978) and the metabolism of phosphorus in vesicular-arbuscular mycorrhizal systems. INTRODUCTION In vesicular-arbuscular (VA) mycorrhizal systems little is known of the physiological processes involved in the development of the fungal infection, or of the active mechanisms involved in the fungal transport of phosphorus (Pearson and Tinker, 1975; Tinker, 1975) and its subsequent transfer from the fungal hyphae into the host cell (Cox and Tinker, 1976). Recently, alkaline phosphatase activity specific to VA mycorrhizas has been reported in onions and tobacco (Gianinazzi-Pearson and Gianinazzi, 1976; Bertheau, 1977). This enzyme activity is closely linked to both the mycorrhizal growth stimulation and the arbuscular phase of the infection (Gianinazzi-Pearson and Gianinazzi, 1978), and there is strong evidence that it is of fungal origin (Gianinazzi- Pearson et al., 1978). Gianinazzi-Pearson and Gianinazzi (1978) have proposed that this VA mycorrhiza-specific alkaline phosphatase could play a role in the assimilation of phosphorus by mycorrhizal roots. However, information concerning its ultrastructural localization is needed if a step is to be made towards understanding the nature of such a role X/79/ $02, The New Phytologist

128 S. GIANINAZZI ETAL. In this paper the ultrastructural distribution of both acid and alkaline phosphatase is examined in onion roots infected by Glomus mosseae (Nicol. & Gerd.

2 128 S. GIANINAZZI ETAL. In this paper the ultrastructural distribution of both acid and alkaline phosphatase is examined in onion roots infected by Glomus mosseae (Nicol. & Gerd.), and the results are discussed in relation to the above hypothesis. MATERIALS AND METHODS Plant material VA mycorrhiza were formed on roots of onions {Allium cepa var. Topaze, F^ hybrid) by infection with G. mosseae as described previously (Gianinazzi-Pearson and Gianinazzi, 1976). Six-week-old onions were used for the cytochemical studies in order to obtain material showing maximum mycorrhiza-specific alkaline phosphatase activity (Gianinazzi-Pearson and Gianinazzi, 1978). Preparation of tissues and enzy^ne methods Three to four mm root segments were cut 2 cm below the onion bulb, fixed immediately at 4 C in 2-5% gluteraldehyde in 0-1 M cacodylate/hcl buffer (ph 7-2) for 2-5 h and then washed consecutively in 5% saccharose/cacodylate buffer, 5% saccharose/hgo and tris-maleate buffer, ph 8-5 (Gomori, 1955). Infected segments were selected under the binocular microscope and hand cut into 0-5 mm sections, the end sections being discarded. Sections were incubated overnight at 4 ^C then 60 min at 37 ""C in the following reaction media. a-naphthyl phosphatase. Na a-naphthyl acid phosphate (4*0 mm) (Sigma) was used as substrate in the presence of 0*12% PbNOg in either 0-05 M acetate buffer, ph 5-0 or tris-maleate buffer, ph 8*5. Controls consisted of sections incubated in the media without substrate, the complete reaction media plus 4 mm KCN, and the complete reaction media plus 20 mm NaE. P'glycerophosphatase. A modified Gomori reaction medium was used which contained 10 mm Na ^-glycerophosphate (Prolabo) in the presence of 0*12% PbNOg in either the acetate or tris-maleate buffer. Control sections were prepared as described above. After incubation sections were rinsed in distilled water, post-fixed for 1 h at 4 C in 2% OSO4 buffered to ph 7*2 with phosphate buffer, and dehydrated through an acetone series. Specimens were embedded in Epon (Epikote) 812 and polymerized at 60 C. Thin sections, cut on a Servall Porter Blum ultramicrotome, were placed on 300 mesh copper-rhodium grids and examined poststained with uranyl acetate using a Zeiss EM 9S2 or a Siemens Elmiskop 102 electron microscope at 60 or 80 kv respectively. Comparative observations were made on 6-week-old roots of non-mycorrhizal onions. RESULTS Enzyme activity was localized by the formation of the black precipitate of the reaction product, lead phosphate. The absence of a precipitate in the no substrate controls (Plate 1, No. 1) showed that any possible confusion between the phosphatase activities and insoluble Ca salts or soluble phosphates in the sections could be eliminated. The ultrastructure of the mycorrhizal fungus and the host cells was typical for this VA mycorrhizal system (see Cox and Sanders, 1974) and changes due to the preparation

3 Localization of phosphatase in mycorrhizas 129 of the specimens were minimal, independent of their treatment. The intercellular hyphae of G. mosseae were thicker walled and contained a large number of discrete vacuoles, separated from each other by their tonoplasts (Plate 3, No. 3). After penetration of the host cell the intracellular hyphae divided dichotomously to form the finer arbuscule branches. The latter were also highly vacuolated (Plate 1, No. 1; Plate 2, Nos 1 and 2) except in their youngest and finest tips, which were identifiable by their small size, little vacuolation and dense cytoplasm (Plate 3, No. 1). As these matured the vacuoles increased in size and number until they almost filled the lumen of the hypha, often leaving only a small peripheral layer of cytoplasm (Plate 1, No. 1). When arbuscules began to senesce, some branches became empty and collapsed (Plate 2, Nos 1 and 2). Nuclei were frequently visible in both arbuscule branches (Plate 2, No. 1) and intercellular hyphae. In nearly all the Plates it can be seen that the intracellular hyphal branches were surrounded by the continuous host plasmalemma and often separated from it by a relatively electron translucent zone. The latter was filled with lighter staining collar material around hyphae near the point of host cell penetration (Plate 3, No. 2). Acid phosphatases In the host cells, NaE inhibited acid /?-glycerophosphatase and acid a-naphthyl phosphatase activities were classically associated with the tonoplast, the endoplasmic reticulum and the dictyosomes (Plate 1, Nos 2 and 3), but their localization within the nucleus, mitochondria and cell wall middle lamella was somewhat irregular. This enzyme distribution was the same in both uninfected onion root cells and those infected by G. mosseae. Acid phosphatase localization in the mycorrhizal fungus depended on the stage of development of the hyphae. In the fine, little vacuolated terminal hyphae of the arbuscule branches, the reaction product precipitate was particularly evident in the dense cytoplasm, the mitochondria and along the tonoplast of the small vacuoles (Plate 1, No. 2). However, similar acid phosphatase activity could not be detected in the more vacuolated, mature arbuscular hyphae (Plate 1, Nos 2 and 3) nor in the collapsed senescent branches. Slight activity was occasionally observ^ed associated with the w^alls of intercellular hyphae but not intracellular hyphae. There was no acid phosphatase activity along the fungal plasmalemma nor in the interface between the fungal wall and the host plasmalemma. Alkaline phosphatases The distribution of alkaline a-naphthyl phosphatase and ^-glycerophosphatase activities within the onion roots was identical. The only difference observed between the two substrates was that ^-glycerophosphate tended to result in a more intense reaction product precipitate than a-naphthyl phosphate (Plate 1, No. 4; Plate 2, No. I). A very weak, rather diffuse alkaline phosphatase activity was present in the cytoplasm of onion cells and this was the same whether they were infected by G. mosseae or uninfected. There was no activity in the interface between the host plasmalemma and the fungal wall in infected cells. On the contrary, conspicuous alkaline phosphatase activity was present in the hyphae of the mycorrhizal fungus. It was always localized within the vacuoles (Plate 2, No. 1) and was never observed along the fungal plasmalemma. This vacuolar activity 3 A NP 82

4 130 S. GIANINAZZI ETAL, was completely inhibited in the presence of 4 mm KCN (Plate 2, No. 2) but was not inhibited by 20 mm NaE (Plate 2, No. 3). It can therefore be concluded that it indeed represented alkaline phosphatase and that it was not acid phosphatase with a large ph spectrum of activity. This, together with the fact that both a-naphthyl phosphate and /?-glycerophosphate served equally well as substrates, suggested that this enzyme activity in the fungal vacuoles could be non-specific alkaline phosphatase. Vacuolar alkaline phosphatase was active in both the intracellular (Plate 2, No. 1) and the intercellular hyphae (Plate 3, No. 3) of G. mosseae. Activity within the intracellular hyphal branches changed with development of the arbuscules. The very young, little vacuolated tips of arbuscule branches showed no activity (Plate 1, No. 4; Plate 3, No. 1). Alkaline phosphatase activity appeared only as the branches became more vacuolated (Plate 3, No. 2), and it increased with their maturation (Plate 1, Nos 4a, b; Plate 2, No. 1). With senescence and collapse of the branches, all enzyme activity disappeared (Plate 1, No. 4b; Plate 2, No. 1). In younger arbuscule branches where vacuolar alkaline phosphatase activity was appearing, it was particularly localized along the fungal tonoplast (Plate 3, No. 2). This distribution became less evident in the vacuoles of mature branches and intercellular hyphae where the phosphatase activities were more intense (Plate 3, No. 3). DISCUSSION Whilst the ultrastructural distribution of acid phosphatases in plant tissues is welldocumented, very little attention has been given to plant alkaline phosphatases. This is particularly true for VA mycorrhizas where ultrastructural studies have been limited to acid phosphatase activities in natural infections (Protsenko, 1973; Scannerini, 1975) in an attempt to find evidence of digestion of the mycorrhizal fungus within host cells. In the present study on the synthesized VA association A. cepa/g. mosseae no indication of such a digestive process has been found, perhaps due to the young state of the mycorrhizal infection. No marked modifications could be detected in the acid phosphatase activities either of the host cells after fungal infection or within the senescing arbuscule branches. In the mycorrhizal fungus, only the contents of the very young, terminal parts of arbuscule branches consistently show considerable acid phosphatase activity and this is never associated with the walls of these intracellular hyphae. As the arbuscule branches become more mature and vacuolation increases, acid phosphatase activity becomes less and less evident. At the same time alkahne phosphatase activity, which is absent from immature hyphae, develops within the fungal vacuoles. When this alkaline phosphatase activity first appears it is fairly weak and seems to be confined to the tonoplast. As the hyphae mature, activity increases and in the well-developed arbuscular and intercellular hyphae, where its activity is particularly intense, its distribution within the vacuoles becomes heterogeneous. With degeneration and collapse of the arbuscule branches all alkaline phosphatase activity disappears. Similar localization of alkaline phosphatase within fungal vacuoles has also been reported for Saccharomyces cerevisiae (Bauer and Sigarlakie, 1975) and Candida albicam (De Nollin, Thone and Borgers, 1975). In the present study, the precise relationship between the vacuolar alkaline phosphatase activity of G. mosseae and the mycorrhiza-specific alkaline phosphatase (MSP) activity detected by Gianinazzi-Pearson and Gianinazzi (1976, 1978) in the same VA mycorrhizal system cannot be defined. However, certain similarities existing between

5 Localization of phosphatase in mycorrhizas 131 the two suggest that they may represent the same enzyme activity. Eirstly, there is now strong evidence that MSP, like the vacuolar activity, is of fungal origin (G. mosseae) (Gianinazzi-Pearson et al.y 1978). Secondly, the properties of the vacuolar phosphatase optimal activity at alkaline ph, inhibition by cyanide, insensitivity to fluoride, hydrolysis of ^-glycerol and a-naphthyl phosphates are also characteristic of MSP. Einally, vacuolar alkaline phosphatase activity increases with maturation of the arbuscular hyphae and therefore the total activity within a mycorrhizal root should depend on the number of active arbuscules present within the infection. Gianinazzi- Pearson and Gianinazzi (1978) have shown that such a correlation exists between the presence of active arbuscules and MSP activity in onion roots infected by G. mosseae. Erom their ultrastructural distribution, it seems probable that the different phosphatases observed in the mycorrhizal fungus are associated with different stages in the development and function of the fungal hyphae. The acid phosphatase activity associated with organelles in the immature tips of arbuscule branches could be involved in the processes of elongation and growth of the hyphae, that is in the development of the infection within the host cells. In light microscope studies, a similar intense acid phosphatase activity has been localized in the actively growing germ tubes developing from spores of G. mosseae (Macdonald and Lewis, 1978). The alkaline phosphatase activity, on the other hand, is localized in the vacuoles of the mycorrhizal fungus and it appears with the development of the hyphae, becoming particularly intense in the mature arbuscular and intercellular hyphae. Polyphosphatelike granules, identified within these same vacuoles of G. mosseae and thought to be involved in the phosphate transport mechanisms, follow a similar distribution pattern within the mycorrhizal infection (Cox et al.y 1975; Tinker, 1975). This correlation, together with the fact that the vacuole is regarded as an active system which probably plays an important role in ion transport mechanisms (Matile and W^iemken, 1976), leads us to think that vacuolar alkaline phosphatase may perhaps be involved in the active mechanism of phosphate transport within hyphae of VA fungi. REFERENCES BAUER, H. & SIGARLAKIE, E. (1975). Localization of alkaline phosphatase in Saccharomyces cerevisiae by means of ultrathin frozen sections. Journal of Ultrastructural Research, 50, 208. BERTHEAU, Y. (1977). Etudes des phosphatases solubles des endomycorhizes ^ v^sicules et arbuscules. D.E.A. thesis, Universite de Dijon, France. Cox, G. & SANDERS, F. (1974). Ultrastructure of the host-fungus interface in a vesicular-arbuscular mycorrhiza. Ne^u Phytologist, 73, 901. Cox, G. & TINKER, P. B. (1976). Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. I. The arbuscule and phosphorus transfer: a quantitative ultrastructural study. Nezv Phytologist, 77, 371. Cox, G. C, SANDERS, F. E. T., TINI^ER, P. B. & WILD, J. (1975). Ultrastructural evidence relating to host-endophyte transfer in a vesicular-arbuscular mycorrhiza. In: Endomycorrhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker) p Academic Press, London and New York. DE NOLLIN, S., THONE, F. & BORGERS, M. (1975). Enzyme cytochemistiyof Candida albicans. Journal of Histochemistry and Cytochemistry, 23, 758. GIANINAZZI-PEARSON, V. & GIANINAZZI, S. (1976). Enzymatic studies on the metabolism of vesiculararbuscular mycorrhiza. I. Effect of mycorrhiza formation and phosphorus nutrition on soluble phosphatase activities in onion roots. Physiologie Vegdtale, 14, 833. GIANINAZZI-PEARSON, V. & GIANINAZZI, S. (1978). Enzymatic studies on the metabolism of vesiculararbuscular mycorrhiza. II. Soluble alkaline phosphatase specific to mycorrhizal infection in onion roots. Physiological Plant Pathology, 12, 45. GIANINAZZI-PEARSON, V., GIANINAZZI, S., DEXHEIMER, J., BERTHEAU, Y. & ASIMI, S. (1978). Les phosphatases alcalines solubles dans l'association endomycorhizienne & v^sicules et arbuscules. Physiologie Vegetale (in press). 5-2

132 S. GIANINAZZI ETAL. GOMORI, G. (1955). Preparation of buffers for use in enzyme studies. In: Methods in Enzymology, vol. i (Ed. by S. P. Colowick & N. O. Kaplan), p. 138.

6 132 S. GIANINAZZI ETAL. GOMORI, G. (1955). Preparation of buffers for use in enzyme studies. In: Methods in Enzymology, vol. i (Ed. by S. P. Colowick & N. O. Kaplan), p Academic Press, London and New York. MATILE, PH. & WIEMKEN, A. (1976). Interactions between cytoplasm and vacuole. In: Transport in plants: III. Encyclop. Plant Physiol. (N.S.), vol. 3, p MACDONALD, R. M. & LEWIS, M. (1978). The occurrence of some acid phosphatases and dehydrogenases in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. New Phytologist, 80, 135. PEARSON, V. & TINKER, P. B. H. (1975). Measurement of phosphorus fluxes in the external hyphae of endomycorrhizas. In: Endomycorrhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker), p Academic Press, London, New York. PROTSENKO, M. A. (1973). Elektronno-mikroscopicheskoye izuchenye Iocalizatie kisloy phosphatzy v perevarivayuschench grib kletkach mikorizy gorocha. Doklady Akademii nauk SSSR, 211, 213. ScANNERiNi, S. (1975). Le ultrastruttore delle micorrize. Giornale Botanico Italiano, 109, 109. TINKER, P. B. H. (1975). Effects of vesicular-arbuscular mycorrhizas on higher plants. In: Symbiosis; Symposium Society Experimental Biology, 29, 325. EXPLANATION OF PLATES Transmission electron micrographs of intracellular and intercellular (Plate 3, No. 3) hyphae of Glomus mosseae in onion roots. Arrows indicate acid or alkaline phosphatase activities. PLATE 1 No. 1. No substrate control. Cross section of a mature arbuscule branch (ab) containing numerous vacuoles (v) and surrounded by the continuous host plasmalemma (p). x No. 2. Acid phosphatase activity (substrate y(?-glycerophosphate) within the cytoplasm and mitochondria (inset) of immature, little vacuolated terminal arbuscule branches (iab). No significant activity is present in the more mature, vacuolated arbuscule branch (ab). In the host cytoplasm some activity is localized along the endoplasmic reticulum (er). x No. 3. Acid phosphatase activity (substrate as No. 2) associated with a dictyosome (d) in the host cytoplasm. The vacuolated arbuscular hypha (ab) shows no activity, x No. 4. Alkaline phosphate activity (substrate as No. 2) witliin the vacuoles of mature arbuscule branches (ab). Activity is absent from the immature, little vacuolated branches (iab) and from the collapsed senescent branches (cab), (a) x 32000; (b) x PLATE 2 No. 1. Alkaline phosphatase activity (substrate a-naphthyl acid phosphate) within the vacuoles of large and fine arbuscule branches (ab). The large hyphal branch contains a nucleus (n). cab, collapsed senescent hyphal branches of arbuscule. x No. 2. KCN control. Alkaline phosphatase reaction (substrate as No. 1) in the presence of 4 mm-kcn. Enzyme activity is completely inhibited and the fungal vacuoles are devoid of the black reaction product precipitate, cab, collapsed senescent arbuscule branch, x No. 3. NaF control. Vacuolar alkaline phosphatase activity (substrate as No. 1) is unaffected by the presence of 20 mm NaF. x PLATE 3 No. 1. Immature, little vacuolated terminal hyphae of arbuscule branches (iab) showing no alkaline phosphatase activity (substrate as Plate 1, No. 2). x No. 2. Vacuolar alkaline phosphatase activity (substrate as Plate 2, No. 1) beginning to appear along the tonoplast (t) in a young arbuscule branch. A collar (c) of host wall material surrounds this branch which is situated near the point of cell penetration, x No. 3. Vacuolar alkaline phosphatase acti\'ity (substrate as Plate 2, No. 1) in an intercellular hypha. No activity is localized along the host cell wall (cw) or plasmalemma (p). x

7 The New Phytologist. Vol. 82, No. 1 Plate 1 4b cab" S. GIANINAZZI ET.11. {Facing p. 132)

8 The Nczu PhvtulosjisI, Vol. 82, No. 1 Plate 2 S. GIANINAZZI

9 The New Phytnh,!^isl, I'ol. 82, No. 1 Plate 3 S. GIANINAZZI I:T.IL.

F.A. SMITH S.E. SMITH

F.A. SMITH S.E. SMITH BIOTROPIA No. 8, 1995: 1-10 NUTRIENT TRANSFER IN VESICULAR-ARBUSCULAR MYCORRHIZAS: A NEW MODEL BASED ON THE DISTRIBUTION OF ATPases ON FUNGAL AND PLANT MEMBRANES*) F.A. SMITH Department of Botany, The