Occurrence of trehalose in vesicular-arbuscular mycorrhizal fungi and in mycorrhizal roots

J. PlantPhysiol. Vol. 140. pp. 41-45 {1992}

Occurrence of Trehalose in Vesicular-Arbuscular Mycorrhizal Fungi and in Mycorrhizal Roots A. 1 2

SCHUBERT!,

P. WYSS2, and A. WIEMKEN2

Istituto Coltivazioni Arboree dell'Universita', V. P. Giuria 15,1-10126 Torino, Italy and Botanisches Institut der Universitat, HebelstraBe 1, CH-4056 Basel, Switzerland

Received June 10, 1991 . Accepted December 9, 1991

Summary

The presence of trehalose was investigated in sporocarps of the vesicular-arbuscular mycorrhizal fungus Glomus versiforme and in roots of Tagetes tenuifolia and Glycine max infected by Glomus mosseae. Trehalose was the main soluble carbohydrate in sporocarps; it was present also in mycorrhizal roots, whereas in nonmycorrhizal roots it was absent or detected only in traces. Soybean plants were inoculated with G. mosseae and grown under different conditions of light regimen and phosphate availability. P fertilization and light deprivation reduced root mycorrhizal infection. The trehalose content of mycorrhizal roots increased concurrently with the course of fungal infection, and decreased both upon P fertilization and light deprivation. The content of total soluble carbohydrates, on the contrary, decreased only upon light deprivation.

Key words: Glomus mosseae, Glomus versiforme, Glycine max, Tagetes tenuifolia, carbohydrate metabolism, plant·microbe interactions. Abbreviations: VAM

=

vesicular-arbuscular mycorrhizae; NMR

=

nuclear magnetic resonance.

Introduction

Vesicular-arbuscular mycorrhizae are symbiotic associations formed between a large number of higher plants on the one side and fungal species of the family Glomaceae on the other. The mutualistic nature of the symbiosis is given by the ability of the fungal partner to take up phosphate and other nutrients from the soil, to translocate them along the extra- and intraradical hyphae to specialized structures called arbuscules and to transfer them from the arbuscules to the host cells (Cooper and Tinker, 1978). Vice versa, carbon compounds are moved from the autotrophic partner to the fungus, where they are used for growth or storage, mainly as lipids, in intraradical vesicles or in newly formed spores (Bevege et aI., 1975; Cooper and Losel, 1978). This mutualistic nutrient exchange is advantageous for plants growing in soils of low nutrient availability, and is of great interest for agricultural application (Hall, 1988). Although the movement of carbon from plant to fungus in the VAM symbiosis has been well demonstrated, little is known about the nature of the molecules that are actually @

1992 by Gustav Fischer Verlag, Stuttgart

exchanged. In analogy with ectomycorrhizal fungi, which readily convert carbon imported from the plant into mannitol and trehalose (Lewis and Harley, 1965; Martin et aI., 1988), an involvement of trehalose in the carbon transfer process was suggested also for VAM (Lewis, 1975). Several authors have found traces of trehalose in spores and extraradical mycelium of VAM fungi (Cooper, 1984; Amijee and Stribley, 1987; Becard et al., 1991), while negative results were reported for mycorrhizal roots (Amijee and Stribley, 1987). In this paper we report further evidence for the occurrence of trehalose in spores of VAM fungi and in mycorrhizal roots of tagetes and soybean, and assess the root trehalose content during the development of mycorrhizae after infection of soybean by the VAM fungus Glomus mosseae.

Material and Methods

Two host plants were used: Glycine max L. Merr. cv. «Maple arrow» and Tagetes tenuifolia Cav.

42

A. SCHUBERT, P. Wyss, and A. WIEMKEN

Tagetes seeds were directly sown in pots containing a steam sterilized (20 min, 121°C) 1: 1 mixture of expanded clay (lecaton R 2-4mm, leca, lamstedt, Germany) and silica sand (Dehne and Backhaus, 1986). Before sowing, pots were either inoculated with spores and infected roots originating from a culture of Glomus mos· seae (Nicol. & Gerd.) Gerdemann & Trappe on Trifolium subterra· neum L., or they were mock-inoculated with non-mycorrhizal roots of T. subterraneum. Plants were watered twice a week with Knop's nutrient solution lacking P. Surface sterilized soybean seeds (5 min in 0.75 % NaCIO) were germinated and the seedlings grown for 4 days in vermiculite. Thereafter, they were transplanted into test plant containers filled with a steam-sterilized mixture of sand, loam and organic matter (3:2: 1), with a pH of 7.6 (Wyss et al., 1991). After a further 4 days the test plant containers were either inoculated by joining them with inoculum containers holding soybean plants with established mycorrhizae of G. mosseae, or mock-inoculated by joining them with containers holding non-mycorrhizal soybean plants (non-inoculated controls). The joined containers were separated by nylon nets (60!lm mesh) across which hyphae, but not roots, could pass. This device, described in detail by Wyss et al. (1991), allowed a fast, reproducible and synchronous infection of the test plant roots. Moreover, individual test plants could be harvested over the infection course without disturbance to the other test plants. Plants were watered daily with the buffered (pH 7) nutrient solution described by Werner and Morschel (1978), containing 5 mM KN0 3 and no P. In the course of the same experiment one series of containers was watered with the same nutrient solution for the first 16 days after inoculation; thereafter, the solution was supplemented with 5 mM P as KH2P0 4 and K2HP0 4 (pH 7). Another series was transferred to the dark 16 days after inoculation. No root nodules were found at the end of the experiment, indicating that no rhizobial infection had taken place. All plants were grown in a glasshouse with natural light; temperature followed a 27°C day/20 °C night cycle, and relative humidity was kept above 60 %. Glomus versiforme (Daniels & Trappe) Berch sporocarps were obtained from 3-month-old leek (Allium porrum L.) plants grown and inoculated as described for tagetes: the epigeous sporocarps were collected and washed free of soil particles and algae before carbohydrate extraction. Microscopic observation revealed no contaminant fungal hyphae inside the sporocarps. For carbohydrate analysis, plant or fungal material was washed, blotted dry, weighed and then extracted twice in 70 % methanol at 65°C (10 mL g-l fresh weight). Purification from polyphenols and desalting were performed following the procedure of Niederer et al. (1989). Before extraction, 100 III 0.1 % D-mannoheptulose was added as internal standard per 1 g fresh weight. Standard solutions of the following sugars and polyols were made: l( + )-arabinose, D( - )-fructose, D( + )-glucose, myo-inositol, D( + )-maltose, D-mannitol, D-pinitol, D( + )-raffinose, D( + )-sucrose and D( + )-trehalose (50 mg dissolved in 1 ml 50 % methanol). These carbohydrates cochromatographed with the main carbohydrates extracted from the tissues; the sum of their contents is referred to in the text as total soluble carbohydrates. The extracts and standards were dried at 60°C under a stream of N2 in gas-chromatography vials. After addition of 3!lg/!ll phenyl/3-glucopyranoside as internal standard, the carbohydrates were converted to their trimethylsilyl oximes and determined by gas liquid chromatography following the method of Keller et al. (1982), using a Shimadzu GC-Mini 3 gas-chromatograph equipped with a flame ionization detector. Columns contained 5% OV-17 Chromosorb W-HP, and nitrogen (50mlmin-l) was used as carrier gas. Peaks were identified by comparison with external standards and, in the case of trehalose, also by the use of internal standards. Root fungal infection was assessed either microscopically after staining (Phillips and Hayman, 1971) or with the grid intersect method (Giovannetti and Mosse, 1980).

All measurements were taken in duplicate and average values are given in the Results section.

Results

Composition of the soluble carbohydrate fraction In the gas-chromatographic analysis of methanol extracts from Glomus versiforme sporocarps a single main peak appeared at the same retention time (18.3 min) as the internal and external trehalose standards (Fig. 1). Thus in sporocarps, trehalose was the main soluble carbohydrate: its concentration was 1.42 mg g-l fresh weight or 94 % of the total soluble carbohydrates. In 3-month-old tagetes plants the content of total soluble carbohydrates was nearly twice as large in mycorrhizal (90 % root infection) than in non-mycorrhizal roots (Table 1). Sucrose, raffinose, glucose and fructose were the prevalent carbohydrates (Fig. 1 and Table 1). Trehalose was present in mycorrhizal roots (4.4 % of total soluble carbohydrates).

7

a A

3

b

9

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34

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V

A

B6

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7

B

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Fig. 1: Profile of gas-chromatographic analysis of soluble carbohydrates from (a) sporocarps of Glomus versiforme, (b) roots of 3month-old, non-inoculated Tagetes tenuifolia, (c) roots of 3-monthold tagetes inoculated with G. mosseae. (A) and (B), internal standards (resp. D-mannoheptulose and phenyl-i3-glucopyranoside); (1), l( + )-arabinose; (2), D-pinitol; (3), D( - )-fructose; (4), D( + )-glucose; (5), myo-inositol; (6), D( + )-sucrose; (7), D( + )-trehalose; (8), D( +)maltose; (9), D( + )-raffinose.

Trehalose in VAM Fungi Table 1: Concentration (mg g-l FW) of soluble carbohydrates in methanol extracts from Glomus versiforme sporocarps and from roots of 3-month-old Tagetes tenuifolia and 4-week-old Glycine max. non-inoculated (C) or inoculated with Glomus mosseae (M). n.d. = not detected (detection limit 1 mgg- 1 FW). G. versiforme

L( + )-arabinose D( - )-fructose D ( + )-glucose myo-inositol D( + )-maltose D-mannitol D-pinitol D( + )-raffinose D( + )-sucrose D( + )-trehalose

sporocarps n.d. n.d. 19.0 8.1 n.d. 9.6 n.d. n.d. 51.7 1423.2

total

1493.9

T. tenuifolia C M 125.8 86.1 211.0 231.2 99.4 166.7 99.2 57.6 10.3 21.4 24.4 48.6 32.0 71.6 337.2 1184.2 746.6 1006.2 n.d. 130.8 1675.3

3025.8

G. max C M n.d. n.d. 81.5 77.6 31.7 40.9 n.d. n.d. n.d. n.d. n.d. n.d. 55.5 67.1 n.d. n.d. 478.9 695.9 n.d. 16.4 697.3

1021.9

Roots of 4-week-old soybean plants (Table 1) contained less soluble carbohydrates than tagetes roots, and the difference between mycorrhizal (80 % root infection) and non-mycorrhizal roots was less than in tagetes. The most abundant carbohydrate was sucrose, followed by fructose, pinitol and glucose. Trehalose was found in mycorrhizal roots, where it had a 1.6 % share of the total soluble carbohydrates.

Changes of trehalose content during the course of mycorrhiza formation and effect of P fertilization and light deprivation Fungal root infection and the concentration of soluble carbohydrates, including trehalose, were measured in soybean roots at time intervals after inoculation with G. mosseae. In inoculated plants, fungal root infection was first detected 10 days after inoculation, then increased up to a nearly steady value above 70 % after 18 days (Fig. 2). In the course of the same experiment, soybean plants with an average 58 % root infection (16 days after inoculation), and noninoculated controls were either fertilized with phosphate or transferred to darkness as described in the Methods section. After an initial further increase, root fungal infection was depressed by both treatments to values below 70 % (Fig. 2). Non-inoculated plants remained non-mycorrhizal until the end of the experiment. After an initial high value followed by a steep drop immediately after inoculation, the content of total soluble carbohydrates in the roots of inoculated plants slowly increased from day 4 after inoculation to day 28 at the end of the experiment. Light deprivation decreased the content of total soluble carbohydrates in the roots of mycorrhizal plants, whereas phosphate fertilization led, after an initial depression, to an increase in the content of soluble carbohydrates (Fig. 3). The content of soluble carbohydrates in the roots of non-inoculated plants was 8 % lower (average of all samples), but followed the same trends as in mycorrhizal roots (data not shown). In inoculated plants, the trehalose content of roots increased starting from day 10, and reached 32 J.l.gg- 1 fresh weight at the end of the experiment (Fig. 4). Upon treatment

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Fig. 2: Changes in percent fungal infection in roots of soybean plants inoculated with G. mosseae and kept in normal conditions (normal light and no P fertilization) along the whole experiment, or kept under normal conditions until day 16 after inoculation and then either transferred to darkness or fertilized with 5 mM P.

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Fig. 3: Changes in the content of total soluble carbohydrates in roots of soybean plants inoculated with G. mosseae and kept under normal conditions (normal light and no P fertilization) along the whole experiment, or kept in normal conditions until day 16 after inoculation and then either transferred to darkness or fertilized with 5 mM P. For symbols compare with Fig. 2.

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Fig. 4: Changes in the content of trehalose in roots of soybean plants inoculated with G. mosseae and kept in normal conditions (normal light and no P fertilization) along the whole experiment, or kept under normal conditions until day 16 after inoculation and then either transferred to darkness or fertilized with 5 mM P. For symbols compare with Fig. 2.

44

A. SCHUBERT, P. WySS, and A. WIEMKEN

with both phosphate fertilization and light deprivation, the trehalose content increased for 2 days, whereafter it either remained constant in the case of P fertilization, or decreased in the case of light deprivation. In both treatments root trehalose content at the end of the experiment was lower than in roots of plants grown in the light without P fertilization. In roots of non-inoculated control plants no trehalose was detected or occasionally only traces, which might be due to the presence of rhizoplane microorganisms.

Discussion Trehalose is widely distributed in microorganisms, particularly in fungi (Elbein, 1974; Thevelein, 1984). Trehalose is also an important component of the soluble carbohydrate fraction of ectomycorrhizae (Lewis and Harley, 1965; Martin et aI., 1988; Niederer et aI., 1989) and of fungi involved in orchid mycorrhizae (Smith, 1967). The presence of trehalose was previously reported in the external mycelium and in spores of VAM fungi (Amijee and Stribley, 1987). Becard et ai. (1991) assessed the presence of trehalose in spores of G. intraradices, G. etunicatum and Gigaspora margarita using l3C NMR. Our results with G. versiforme sporocarps confirm these findings and show that trehalose is the most important soluble carbohydrate in these VAM propagules. The importance of trehalose as a reserve has been suggested in those cases where fungal propagules germinate on nutrient-poor media, as in the case of Neurospora conidia (Sussman and Douthit, 1973). Spores of VAM fungi also germinate in nutrient-poor media (Daniels and Graham, 1976)_ Until now, trehalose could not be demonstrated in roots inoculated with VAM fungi, although several attempts were made (Hayman, 1974; Bevege et aI., 1975; Amijee and Stribley, 1987)_ Our results show that trehalose is present in VAM roots of tagetes and soybean. Furthermore, the trehalose content in mycorrhizal roots changes concurrently with the degree,of root fungal infection, both when the latter increases after inoculation and when it is artificially depressed by fertilization or light deprivation. In mycorrhizal plants deprived of light, the content of trehalose and of other soluble carbohydrates in the roots decreased concurrently. An interpretation of this pattern is that trehalose is produced in the fungus with carbon supplied by the plant. In the mycelium of an ectomycorrhizal symbiont, Martin et al. (1988) showed that newly gained carbon is first incorporated into mannitol and then directed to trehalose synthesis. We found only trace amounts of mannitol in spores and in the intraradical mycelium, and negative results have been reported before (Hayman, 1974; Bevege et aI., 1975)_ Thus, in the case of VAM fungi, assimilated carbon transferred from the plant might be incorporated into trehalose without involvement of mannitol. The trehalose content of mycorrhizal soybean roots was reduced by P fertilization. This decrease in the trehalose content may be explained by the decrease in root fungal infection. However, an additional hypothesis can be made: it has been shown that an ample P supply decreases the exudation of assimilated carbon from the root cells (Ratnayake et aI.,

1978). Thus in our experiment, P fertilization might have decreased the amount of assimilated carbon released by the root cells and available for fungal synthesis of trehalose. Acknowledgements

Research supported by the National Research Council of Italy, Special Project RAISA, Subproject 2, paper n. 297, and by the Swiss National Science Foundation. The authors wish to thank Prof. Thomas Boller for critical reading of the manuscript and Kurt In~ichen for technical assistance in the gas-chromatographic analYSIS.

References AMIJEE, F. and P. STRIBLEY: Soluble carbohydrates of vesicular-arbuscular mycorrhizal fungi. The Mycologist 3,21-22 (1987). BEcARD, G., L. W. DONER, D. B. ROLIN, D. D. DOUDS, and P. E. PFEFFER: Identification and quantification of trehalose in vesicular-arbuscular fungi by in vivo 13C NMR and HPLC analyses. New Phytol. 118, 547-552(1991). BEVEGE, D. 1., G. D. BOWEN, and M. F. SKINNER: Comparative carbohydrate physiology of ecto- and endomycorrhizas. In: SANDERS, F. E., B. MOSSE, and P. B. TINKER (eds.): Endomycorrhizas, p. 149-174. Academic Press, London (1975). COOPER, K. M.: Physiology of VA mycorrhizal associations. In: PoWELL, C. LL. and D. J. BAGYARAJ (eds.): VA mycorrhiza, p. 155-186. CRC Press. Boca Raton, USA (1984). COOPER, K. M. and D. M. LOSEL: Lipid physiology of vesicular-arbuscular mycorrhiza. 1. Composition of lipids in roots of onion, clover and ryegrass infected with Glomus mosseae. New Phytol. 80,143-151 (1978).

COOPER, K. M. and P. B. TINKER: Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. II. Uptake and translocation of phosphorus, zinc and sulphur. New Phytol. 81, 43 -52 (1978). DANIELS, B. A. and S. O. GRAHAM: Effects of nutrition and soil extracts on germination of Glomus mosseae spores. Mycologia 68, 108 -116 (1976). DEHNE, H.-W. and G. F. BACKHAUS: The use of vesicular-arbuscular mycorrhizal fungi in plant production. I. Inoculum production. J. Plant Diseases and Protection 93, 415-424 (1986). ELBEIN, A. D.: The metabolism of a,a-trehalose. Adv. Carbohydrate Chern. Biochem. 30, 227 - 256 (1974). GIOVANNETTI, M. and B. MOSSE: An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytol. 84, 489-500 (1980). • fuLL, I. R.: Potential for exploiting vesicular-arbuscular mycorrhizas in agriculture. Advances in Biotechnological Processes 9, 141-171 (1988). HAYMAN, D. S.: Plant growth responses to vesicular-arbuscular mycorrhiza. VI. Effects of light and temperature. New Phytol. 73, 71-80 (1974). KELLER, F., M. SCHELLENBERG, and A. WIEMKEN: Localization of trehalose in vacuoles and of trehalose in the cytosol of yeast (Saccharomyces cerevisiae). Arch. Microbiol. 131, 298-301 (1982). LEWIS, D. H.: Comparative aspects of the carbon nutrition of mycorrhizas. In: SANDERS, F. E., B. MOSSE, and P. B. TINKER (eds.): Endomycorrhizas, p. 119-148. Academic Press, London (1975). LEWIS, D. H. and J. L. HARLEY: Carbohydrate physiology of mycorrhizal roots of beech. 1. Identity of endogenous sugars and utilization of exogenous sugars. New Phytol. 64, 224-237 (1965). MARTIN, F., M. RAMSTEDT, K. SODERHALL, and D. CANET: Carbohydrate and amino acid metabolism in the ectomycorrhizal as-

Trehalose in V AM Fungi comycete Sphaerosporella brunnea during glucose utilization: a carbon-13 NMR study. Plant Physiol. 86, 935-940 (1988). NIEDERER, M., W. PANKOW, and A. WIEMKEN: Trehalose synthesis in mycorrhizae of Norway spruce: an indicator of vitality. Eur. J. For. Pathol. 19, 14-20 (1989). PHILLIPS, J. M. and D. S. HAYMAN: Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. mycol. Soc. 55, 158-160 (1971). RATNAYAKE, M., R. T. LEONARD, and J. A. MENGE: Root exudation in relation to supply of phosphorus and its possible relevance to mycorrhizal formation. New Phytol. 81, 543-552 (1978).

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SMITH, S. E.: Carbohydrate translocation in orchid mycorrhizas. New Phytol. 66, 371-378 (1967). SUSSMAN, A. S. and H. A. DOUTHIT: Dormancy in microbial spores. Annu. Rev. Plant Physiology 24,311-352 (1973). THEVELEIN, J. M.: Regulation of trehalose mobilization in fungi. Microbiol. Rev. 48, 42-59 (1984). WERNER, D. and E. MCiRSCHEL: Differentiation of nodules of Glycine max. Ultrastructural studies of plant cells and bacteroids. Planta 141, 169-177 (1978). WYSS, P., T. BOLLER, and A. WIEMKEN: Phytoalexin response of soybean roots to separate or combined infections with a mycorrhizal fungus and a pathogen. Experientia 37, 395-399 (1991).