MANNITOL DOES NOT INHIBIT GLYCOLYTIC ENZYMES IN ROOTS OF PINUS S YL VESTRIS AND FAGUS ORIENTALIS

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1 New Phytol. (1986) 102, MANNITOL DOES NOT INHIBIT GLYCOLYTIC ENZYMES IN ROOTS OF PINUS S YL VESTRIS AND FAGUS ORIENTALIS BY RAIDA JIRJIS*, MAURITZ RAMSTEDT AND KENNETH SODERHALLf Institute of Physiological Botany, University of Uppsala, Box 540, S Uppsala, Sweden {Accepted 14 October 1985) SUMMARY Glucose-6-phosphate dehydrogenase, hexokinase and phosphoglucose-isomerase extracted from roots of Pinus sylvestris L. or Fagus orientalis Lipsky were not inhibited by mannitol at concentrations up to 05 M in contrast to previous reports for these enzymes from Fagus sylvatica L. The K^n^-values for the carbohydrate substrates were the same in the presence or absence of mannitol (50 nriivi). Key words: Mannitol, enzyme inhibition, glycolytic enzymes, mycorrhiza, roots. INTRODUCTION Harley and co-workers have made significant contributions to our understanding of biochemical regulaton in beech mycorrhizas (Lewis & Harley, 1965a-c; Wedding & Harley, 1976; Harley, 1978; Harley & McCready, 1981; Warren, Wilson & Harley, 1983). Lewis & Harley (1965c) put forward the hypothesis that mannitol may exert a regulatory role in this symbiosis. Briefly, the fungal symbiont depletes the host roots of soluble carbohydrate and converts it to mannitol and/or trehalose which are less metabolically accessible to cells in the host root. Wedding & Harley (1976) suggested that mannitol is translocated from the fungus into the cytoplasm of host roots where it inhibits certain glycolytic enzymes. This could result in an increase in the amount of glucose in the host root. Since Wedding & Harley (1976) reported that mannitol inhibited several glycolytic enzymes of beech roots, we have tested whether it could similarly inhibit those of Pinus sylvestris L. We also included extracts of non-mycorrhizal roots of Fagus orientalis Lipsky in our investigation to compare our results with those of Wedding & Harley (1976). MATERIALS AND METHODS Plants Roots were collected from about four week-old seedlings of P. sylvestris which had been grown on peat or vermiculite and incubated at 20 C in continuously illuminated chambers. Beech roots {F. orientalis) were taken from about six * Present address: Department of Forest Products, Swedish University of Agricultural Sciences, Box 7008, S Uppsala, Sweden. t To whom correspondence should be addressed X/86/ $03.00/ The New Phytologist

2 286 R. JiRjis e«al. month-old seedlings grown outdoors. Only root tips and white parts of the roots were collected. These were washed thoroughly and either used immediately or lyophilized and stored at 20 C. Preparation of cell free extracts The roots were ground in an ice-cold mortar with 4 % (w: v) polyvinylpolypyrrolidone (PVPP) in 0-1 M potassium phosphate buffer, ph 6-8, containing 1 mm EDTA, 0-5 mm phenylmethylsulfonyl fluoride and 1 mm mercaptoethanol, and then further homogenized in a glass piston homogenizer. The homogenate was first centrifuged at 2000 g (10 min, 4 C) to remove cell debris and subsequently at g for 20 min at 4 C. The PVPP used was insoluble, which enabled it to be precipitated during centrifugation. No interference from phenolic compounds or phenoloxidase activity was observed in the extracts, since the extracts showed no signs of browning during experiments and no loss of enzyme activity was observed during prolonged storage (^12h) at 6 C. The supernatant from extracts of beech was dialysed against the extraction buffer for 16 h. For the pine extract, the dialysis step was replaced by desalting the supernatant on a Sephadex G-25 column (PD-10, Pharmacia, Sweden) to remove endogeneous substrate such as 6-phosphogluconate which interferes with the reduction of NADP by glucose 6-P-dehydrogenase in unpurified extracts. The extracts were then used without further treatment. The temperature of the extracts was maintained at 2 to 4 C during all stages until assayed. Enzyme assays General. Spectrophotometric assays were carried out at 25 C (1 cm light path) using a Hitachi spectrophotometer. In a final reaction volume of 1 ml, ii\ of the enzyme extract was used. Enzyme activities were determined by measuring the increase in absorbancy at 340 nm caused by reduction of NADP. Protein determinations were made following the method of Lowry et al. (1951) using bovine serum albumine as standard. For all enzymes controls were run with reaction mixtures lacking substrate, cofactor, fungal extract or commercial enzyme, respectively. For phosphoglucose isomerase, fructose 6-phosphate was preincubated with the commercial glucose 6-phosphate dehydrogenase for a few minutes to remove contaminant glucose 6-phosphate from the substrate. When studying the effect of mannitol on the X^ for the enzyme substrates, the mannitol concentration was varied between 0 and 50 mm. However, enzyme activity was tested at concentrations up to 0-5 M mannitol. Assay mixtures of the different enzymes were as follows: Glucose 6-phosphate dehydrogenase {EC ). 10 mm MgClj, 0 5 mm NADP in 20 mm HEPES buffer ph 7 5. The concentration of glucose 6-phosphate was varied between 0.03 to 1 mm. Hexokinase {EC ). Using glucose as substrate, activity was determined with a coupled assay; 10 mm MgClj, 0 5 mm NADP, 1 mm ATP, 0 5 IU glucose 6-phosphate dehydrogenase in 20 mm HEPES buffer ph 7.2. Glucose concentrations from to 1 mm were used. Hexokinase with fructose as substrate was assayed in a similar manner, adding 0 5 IU phosphoglucose isomerase. Fructose concentration was varied between 0 25 and 5 0 mm.

3 Mannitol and glycolytic enzymes of roots 287 Phosphoglucose isomerase {FC ). 10 mm MgClj, 05 mm NADP, 0-5 IU glucose 6-phosphate dehydrogenase in 20 mm HEPES buffer ph 7 5. Substrate concentration was varied between 0125 and 10 mm fructose 6-phosphate. Chemicals All commercial enzymes, NADP, substrates, HEPES and EDTA were purchased from Sigma Chemical Co. Sephadex G 25 was from Pharmacia Fine Chemicals, Inc. All other chemicals were of the highest analytical grade commercially available. RESULTS AND DISCUSSION The Kj^ values for the different enzymes, determined by means of Lineweaver-Burk plots (Fig. 1) were similar for extracts of both pine and beech roots except for hexokinase with fructose as substrate where pine had a tenfold higher K^ (Table 1). The affinity of the substrates for their respective enzymes was not altered for any of the four enzymes from pine or beech with concentrations of mannitol as high as 50 mm (Fig. 1). Furthmore, no effect on I^^x ^^^ found even at the highest concentration tested (0-5 M) (data not shown). V^^^ for the different enzymes varied between the different preparations probably owing to the condition of the roots. However, the /C^-values were the same regardless of preparation. A commercial preparation of yeast glucose 6-phosphate dehydrogenase was included for comparison and mannitol, at a concentration of 50 mm, did not alter the K^ value for glucose 6-phosphate or the I^ax f ^^^ raction. The K^ values of the enzymes tested were similar to those of the yeast, Saccharomyces cerevisiae, reported by Barman (1969) (Table l).the K^ (glucose 6-phosphate) of glucose 6-phosphate dehydrogenase from pine and beech were also similar to that reported for this enzyme from roots of F. sylvatica by Wedding & Harley (1976). The K.^ values calculated for the two other enzymes, hexokinase and phosphoglucose isomerase, however, were different from those reported for the corresponding enzymes from this source (Table 1). The reason for this difference between enzymes extracted from roots of two closely related species is not known. Table 2 shows that relatively high concentrations of mannitol, up to 50 mm, did not have any eftect on glucose 6-phosphate dehydrogenase or hexokinase extracted from pine and beech. Since we removed 6-phosphogIuconate from the extracts, no substrate for 6-phosphogluconate dehydrogenase was present and no substrate could be produced if G6PDH was inhibited by mannitol. Therefore, the failure of mannitol to affect NADP-reduction in the root extracts with glucose 6-P and NADP as substrates clearly shows that mannitol does not inhibit glucose 6-P dehydrogenase. Wedding & Harley (1976), however, reported inhibition of enzyme activity of glucose 6-phosphate dehydrogenase and hexokinase, using mannitol at concentrations which they thought likely to be present in the root cells. Earlier reports on the effects of mannitol and other polyols on enzymes mostly deal with concentrations in the range of 1-5 to 3-0 M (Ruwart & Suelter, 1971; Bradbury & Jacoby, 1972; Damjanovichefa/., 1972; Myers &Jacoby, 1973). Such concentrations of polyols, which are certainly above the physiological level within the cell, can either inhibit or enhance the activity of several enzymes. For example in yeast, glucose 6-phosphate dehydrogenase activity is inhibited (Myers & Jacoby, 1973) whereas pyruvate kinase activity is enhanced (Ruwart & Suelter, 1971).

4 288 R. JiRjis et al. ( a ) 0-6r PGI GK 0-28r F6P (mm-') Glucose (mm"') FK Glucose (mm"') 0-6r G6PDH Fructose (mm"') 80 Fig. 1. Lineweaver-Burk plots of (a) pine root extract and (b) beech root extract. Solid symbols: 0 nnm mannitol, open symbols 30 mm mannitol. The activities with and without mannitol added to the incubation mixture were the same. Enzyme extraction and assay as in 'Material and Methods'. Abbreviations: PGI, phosphoglucose isomerase; GK, hexokinase (glucose); FK, hexokinase (fructose); G6PDH, glucose 6-phosphate dehydrogenase; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate.

5 Mannitol and glycolytic enzymes of roots 289 Table 1. Michaelis constants for four enzymes extracted from different organisms Enzyme Present work Pine Beech m (mm) Reported Yeast data Beech Glucose 6-phosphate-dehydrogenase Hexokinase (glucose) Hexokinase (fructose) Phosphoglucose isomerase on ' ND Barman (1979); fwedding & Harley (1976); ND, not determined. K^ values (average of 2 to 4 separate experinients) were determined using Lineweaver-Burk plots. Table 2. Fffect of mannitol on different enzymes extracted from pine and beech Mannitol concentration (mm) Enzymes Suhstrate Pine 0-50 Beech 0-50 Beech* Glucose 6-phosphate-dehydrogenase Hexokinase Hexokinase Phosphoglucose isomerase G6P Glucose Fructose F6P I (018) I (0-27) I (0-20) NI I, inhibition; NI, No inhibition. Data from Wedding & Harley (1976). The range of mannitol concentrations used was not given and could not be deduced from the data reported, ^i values (mm) for inhibition by mannitol in parentheses. If the proposals of Lewis & Harley (i965a) and Wedding & Harley (1976) that the concentration of mannitol in mycorrhizal roots of beech is in the millimolar range are correct, then the fungus is unlikely to provide the root with sufficient mannitol to exert any effect on enzymes of the glycolytic pathway. Based upon their results. Wedding & Harley (1976) proposed that soluble carbohydrates would be made more available to the fungus owing to the inhibitory effect of mannitol on enzymes of the glycoltyic pathway. As our results do not support this hypothesis, we, therefore, conclude that mannitol in mycorrhizal associations in P. sylvestris and F. orientalis has no inhibitory effect on the glycolytic enzymes and so will not result in enhanced levels of glucose within the root cytoplasm. It is generally agreed that the formation of mycorrhiza requires high quantities of soluble sugars in the cell to be infected (Harley, 1969). Lewis & Harley (1965c) have proposed that the fungal symbiont depletes the host root of soluble sugars (glucose and/or fructose) and forms a gradient by transforming these soluble sugars into mannitol and/or trehalose. Therefore, it should be instructive to compare the rate of glycolysis occurring in the root cytoplasm with the amount of glucose/fructose translocated into the fungal cytoplasm. Presently, we cannot devise any experiments to assay this but they are urgently needed so that the metabolism and translocation of carbohydrates in the symbiotic system can be understood. Piche, Fortin & Lafontaine (1981) showed, in a study on pine roots, that the cortical cells of both mycorrhizal and non-mycorrhizal short roots lacked

6 29O R. JiRjis ef al. polysaccharide inclusions which were abundant in the cortex of long roots. Since it is mostly the short roots that take part in the mycorrhizal association, this implies that soluble sugars are available in the short roots prior to the initiation of mycorrhiza. Furthermore, the data of Piche et al. (1981) indicate that the rate of polysaccharide synthesis in short roots is low or even zero, or alternatively that a very rapid turnover of polysaccharides occurs. Therefore, the amount of soluble sugars, such as glucose and fructose, may he high enough both to provide substrate for glycolysis in the host root cells and to support the mycorrhizal symbiont. It is well established that roots exude, among many other substances, soluble carbohydrates (Scott Russell, 1977; Schwab, Leonard and Menge, 1984) and, hence, these substances may be available for the mycorrhizal symbiont which, via the mannitol cycle, (Ramstedt, Niehaus & Soderhall, 1986) could transform glucose or fructose into mannitol, a sugar alcohol that cannot be metabolized by the host. Regulation of the mannitol metabolism may therefore be of the utmost importance in the balance of sugar uptake from the host. This requires that the host roots cannot metabolize mannitol or that significant amounts of mannitol are not able to diffuse into the host cells. Preliminary experiments show that pine roots have neither mannitol 1-phosphate dehydrogenase nor mannitol dehydrogenase (Ramstedt & Soderhall, unpublished). The fungus can in this way maintain a diffusion gradient, as proposed by Lewis & Harley (1965c), without having to interfere with glycolytic metabolism in the host for uptake of carbohydrates. In this context, high levels of soluble carbohydrates would not be caused by the fungal infection, but be a prerequisite for mycorrhizal formation, partly determining compatibility or non-compatibility of the roots. ACKNOWLEDGEMENTS We thank Prof. R. T. Wedding and Prof. J. L. Harley for reading our manuscript. This work has been supported by grants from the Swedish Agricultural Research Science Council (S93:2) and the Swedish Natural Science Research Council. REFERENCES BARMAN, T, E. (1969), Enzyme Handbook, vols I & ii, Springer-Verlag, New York, BRADBURY, S, L, & JACOBY, W, B, (]972), Glycero) as an enzyme-stabilizing agent: effects on aldehyde dehydrogenase. Proceedings of the National Academy of Sciences, 69, DAMJANOVIC, S., BOT, J., SOMOGYI, B, & SUMEGI, J, (1972), Effect of glycerol on some kinetic parameters of phosphorylase b, Biochimica et Biophysica Acta, 284, , HARLEY, J, L, (1969), The Biology of Mycorrhiza, 2nd Edn, Leonard Hill, London, HARLEY, J, L. (1978), Ectomycorrhizas as nutrient absorbing organs. Proceedings of the Royal Society, London, B, 203,1-21, HARLEY, ], L, & MCCREADY, C, C, (1981), Phosphate accumulation in Fagus mycorrhizas. New Phytologist, 89, LEWIS, D, H. & HARLEY, J, L, (1965a), Carbohydrate physiology of mycorrhizal roots of beech 1, Identity of endogenous sugars and utilization of exogenous sugars. New Phytotogisl, 64, , LEWIS, D, H, & HARLEY, J. L, (1965b), Carbohydrate physiology of mycorrhizal roots of beech, II, Utilization of exogenous sugars by uninfected and mycorrhizal roots. New Phytologist, 64, , LEWIS, D, H, & HARLEY, J, L, (]965c). Carbohydrate physiology of mycorrhizal roots of beech, HI, Movement of sugars between host and fungus. New Phytologist, 64, , LowRY, O. H., RosEBROUGH, N, J,, EARR, A, L, & RANDALL, R, J, (1951), Protein measurement with the Eolin phenol reagent. Journal of Biological Chemistry, 193, , MYERS, ], S, & JACOBY, W, B, (1973), Effect of polyhydric alcohols on kinetic parameters of enzymes. Biochemical and Biophysical Re.search Communications, 51, ,

7 Mannitol and glycolytic enzymes of roots 291 PiCHE, Y., FoRTiN, J. A. & LAFONTAINE, J. G. (1981). Cytoplasmic phenols and polysaccharides in ectomycorrhizal and non-mycorrhizal short roots of pine. Nezii Phytologist, 88, RAMSTEDT, M., NIEHAUS, W. G. & SODERHALL, K. (1986). Mannitol metabolism in the mycorrhizal fungus Piloderma croceum. Experimental Mycology (in press). Ru'WART, M. J. & SuELTER, C. H. (1971). Activation of yeast pyruvate kinase by natural and artificial cryoprotectants. The Journal of Biological Chemistry, 246, SCHWAB, S. M., LEONARD, R. T. & MENGE, J. A. (1984). Quantitative and qualitative comparison of root exudates of mycorrhizal and nonmycorrhizal plant species. Canadian Journal of Botany, 62, SCOTT RUSSELL, R. (1977). Plant Root Systems. McGraw Hill, Maidenhead. WARREN WILSON, J. & HARLEY, J. L. (1983). The development of mycorrhiza on seedlings of Fagus sylvatica L. New Phytologist, 95, WEDDING, R. T. & HARLEY, J. L. (1976). Fungal polyol metabolites in the control of carbohydrate metabolism of mycorrhizal roots of beech. New Phytologist, 77,

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