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Phosphohexoisomerase in Beech Mycorrhiza Author(s): J. L. Harley and B. C. Loughman Source: New Phytologist, Vol. 65, No. 2 (Apr., 1966), pp. 157-160 Published by: Wiley on behalf of the New Phytologist Trust Stable URL: http://www.jstor.org/stable/2430354 . Accessed: 14/06/2014 21:14 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to New Phytologist. http://www.jstor.org This content downloaded from 195.78.109.66 on Sat, 14 Jun 2014 21:14:15 PM All use subject to JSTOR Terms and Conditions

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Page 1: Phosphohexoisomerase in Beech Mycorrhiza

Phosphohexoisomerase in Beech MycorrhizaAuthor(s): J. L. Harley and B. C. LoughmanSource: New Phytologist, Vol. 65, No. 2 (Apr., 1966), pp. 157-160Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2430354 .

Accessed: 14/06/2014 21:14

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to NewPhytologist.

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Page 2: Phosphohexoisomerase in Beech Mycorrhiza

PHOSPHOHEXOISOMERASE IN BEECH MYCORRHIZA

BY J. L. HARLEY* AND B. C. LOUGHMAN

Department of Agriculture, Oxford

(Received 23 July I965)

SUMMARY

Phosphohexoisomerase is shown to be present in beech mycorrhizas and to be distributed between host and fungal tissues approximately in proportion to dry weight. These results show that the different destinations of glucose and fructose absorbed by mycorrhizas is not explicable in terms of the absence of this enzyme from mycorrhizas or from their fungal tissues.

INTRODUCTION

In their study of carbohydrate physiology of beech mycorrhiza, Lewis and Harley (i965a, b, c) observed that externally supplied glucose was absorbed and mainly assimi- lated in the sheath tissue into trehalose and glycogen. By contrast, absorbed fructose was mainly assimilated into mannitol. The behaviour of sucrose was intermediate, both mannitol and the polymers of glucose being usually formed. From this it was concluded that one possibility was that the activity of phosphohexoisomerase might be low com- pared with the rates of phosphoglucomutase on the one hand and mannitol phosphate dehydrogenase on the other (Lewis and Harley, i965b). Uninfected beech tissue and the core tissue of mycorrhizas (stripped of the fungal sheath) rapidly synthesized sucrose from either glucose or fructose and produced labelled glucose when absorbing labelled fructose and vice versa.

In this paper an investigation of the phosphohexoisomerase activity of extracts of mycorrhizal tissue is made.

METHOD

Samples of plant material were ground in ice-cold 0.02 M tris buffer (pH 8.o) in a chilled mortar. The homogenate was spun at 3000 rev/min for io minutes and the supernatant was decanted.

The enzyme activity was assayed over measured periods of time in a digest consisting of 0.4 ml glucose-6-P (40 mg/ml), 0.5 ml of the supernatant, 0.4 ml H20. For each sample 0.2 ml was withdrawn for fructose assay. The fructose reagent consisted of a mixture of two solutions: (I) 200 ml concentrated HCI containing 42 mg CUS04.5 H20 per litre, 260 ml A.R. glycerol and ioo ml H20; and (2) resorcinol 0.45 g/Ioo ml. The solutions were kept cool in dark bottles and mixed fresh each day as follows: acid reagent IOO ml, resorcinol 20 ml, water 36 ml.

Exactly 0.2 ml of test solution was added to 7.8 ml of the fructose reagent in a test tube. After thorough mixing, the tubes were heated for exactly 12 minutes in a boiling water bath and cooled in running water. The intensity of the colour produced was read in the Spekker absorptiometer using an Ilford 6oi violet filter against a reagent blank. The quantities of fructose were estimated using a calibration curve.

* Present adress: Department of Botany, The University, Sheffield. '57

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Page 3: Phosphohexoisomerase in Beech Mycorrhiza

I58 J. L. HARLEY AND B. C. LOUGHMAN

It was found that an inhibitor of the enzyme occurred in mycorrhizal roots. Since this might well be associated with the tannins and phenolic compounds known to be present, extracts were made after the tissue had been injected under reduced pressure with 5% (w/v) polyvinylpyrrolidone (PVP, Kollidone z5, Victor Blagden & Co.) In some cases, I% PVP was also added to the extracting fluid. This method was a modification of that described by Hulme and Jones (I962) for preparation of mitochondria.

Plant material The plant material used consisted of i cm tips of beech mycorrhizas sampled and

washed as formerly described (Harley and McCready, I952). For certain experiments these were dissected into central core and sheath components and the separated tissue utilized in experiments. In other cases whole barley root systems of 6-day seedlings were extracted for comparison.

200

160 0

au) 120 -

L / t /

?2 80 -

40/=

30 60 90 120 Time (minutes)

Fig. I. The effect of the extract of beech mycorrhizas upon the activity of phosphohexo- isomerase of barley roots. 750 mg of 6-day-old barley roots extracted with 5.0 ml of tris buffer pH 8.o with ( x ) and without (@) 250 mg of beech mycorrhizas. Each digest contained the extract of 75 mg barley roots with or without that of 25 mg of beech mycorrhizas.

RESULTS

In a first experiment, aliquots of extract equivalent to five mycorrhizal tips gave no significant phosphohexoisomerase activity over periods of time up to 4 hours, whether or not dithionite (5 mg/5 ml) was added to the tris buffer during extraction.

Extracts were therefore made of barley root systems and of barley and beech mycor- rhizas together. The results in Fig. i show that the isomerase activity of barley was completely inhibited by beech extract over periods of I20 minutes. It follows therefore that in the beech mycorrhizas there is an inhibitor of the enzyme, a state of affairs similar to that investigated by Harley and ap Rees (I959) who studied cytochrome oxidase activity of the beech mycorrhizas.

Since Hulme and Jones (i962) have reported that polyvinylpyrrolidone can be used to protect mitochondrial preparations against phenolic compounds during extraction, a modification of their method was used. In Table i it is clear that the inhibition of the

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Page 4: Phosphohexoisomerase in Beech Mycorrhiza

PPhosphohexoisomerase in mycorrhiza I59

barley enzyme by beech mycorrhizal extract is confirmed. But when the mycorrhizas were first infiltrated with PVP, the preparation showed greater activity than the barley alone and a great part of this must have been derived from the mycorrhizal tissues.

Table i. Phosphohexoisomerase in beech mycorrhiza and barley roots Fructose (,ug/0.2 ml)

Enzyme I minute i 5 minutes 45 minutes i. Barley extracted in tris 14 25 37.5 2. Barley+beech extracted in tris 24 26 29 3. Barley+beech extracted tris+PVP 38 8i i77 4. Beech extracted in tris 41.5 51 51 5. Beech extracted in tris+PVP 74 128 235.5

Sample i. Extract of I g of barley roots in 5 ml tris buffer. Sample 2. Extract of barley (i g) and beech (0.25 g) in 5 ml tris buffer. Sample 3. The same extracted in tris buffer + I % PVP (5 ml), the beech mycor-

rhizas previously infiltrated with 5 % PVP. Sample 4. Tris extract of 0.5 g beech mycorrhizas. Sample 5. Tris + I % PVP extract of o.5 g beech mycorrhizas infiltrated with

5% PVP. Digest: i6 mg glucose-6-P, 0.5 ml enzyme. Total volume 1.3 ml.

Results of extracting beech mycorrhizas alone are given in Table 2. The extract made in the presence of PVP showed high activity as opposed to the negligible activity of the control without PVP. Boiling destroyed the enzyme but the presence of PVP in excess in the digest did not diminish activity.

Table 2. Phosphohexoisomerase in beech mycorrhiza Sample Enzyme Additions Fructose (Pg/0.2 ml)

to digest I minute 20 minutes 45 minutes I Tris extract A - 32.8 35.0 66.9 2 Tris+PVP extract B - 49.3 123.0 173.6 3 Tris+PVP extract B boiled 49.3 49.3 46.o 4 Tris + PVP extract B 0.4% PVP 52.5 120.8 155.0

Samples of 0.5 g fresh beech mycorrhiza (sixty tips) extracted as follows: A, 5 ml tris buffer; B infiltrated with 5 % PVP extracted in 5 ml tris buffer containing I % PVP. Each digest contained i6 mg glucose-6-P + 0.5 ml enzyme. Total volume .3 ml.

These results confirm the presence of phosphohexoisomerase in mycorrhizal tissue. It is now necessary to demonstrate its presence in the fungal sheath for it is here that absorbed glucose and fructose did not appear to be readily interconvertible in the experiments of Lewis and Harley (i965a, b, c).

Table 3. Fructose produced with glucose-6-P by phosphohexoisomerase activity of extracts of the sheath and core tissues of twenty-five mycorrhizal

roots Time Fructose produced (pg) Activity in

(minutes) Core Sheath Total sheath (%) I5 I6.5 11.5 28.o 41 30 21.0 I8.o 39.0 46 45 35.5 26.o 6i.5 42 6o 46.5 37.0 83.5 44

Dissections after infiltration with 5% PVP. Extraction and digest as Table 2 (B).

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Page 5: Phosphohexoisomerase in Beech Mycorrhiza

i6o J. L. HARLEY AND B. C. LOUGHMAN

Table 3 shows the activity of the separate sheath and core tissues of beech mycorrhizas dissected before extraction in tris buffer. A little over 40% of the sum of the activity of the two tissues was extracted from the sheath. It has been shown previously (Harley and McCready, 1952) that the sheath comprises approximately 39% of the dry weight of the mycorrhizal root-tip. Hence the phosphohexoisomerase activity appears to be equally distributed on a dry weight basis in the constituent tissues of mycorrhizas.

It is clear from these experiments that the suggestion made by Lewis and Harley (i965b) that phosphohexoisomerase activity might be deficient in beech mycorrhizas and particularly in their sheath tissues is groundless. The explanation of the different destinations of absorbed glucose and fructose in the sheath tissue must be found else- where. Not only are the relative rates in vivo and in vitro of phosphoglucomutase, mannitol phosphate dehydrogenase and phosphohexoisomerase worth examination, but the pathway of synthesis of mannitol should receive attention. For if the polyol were synthesized directly from free fructose the rate-limiting step in the conversion of glucose to mannitol might be a specific fructose-6-phosphatase and not phosphohexoisomerase. Conversion of fructose to glucose polymers would then depend on relative activities of fructokinase and mannitol dehydrogenase.

REFERENCES

HARLEY, J. L. & AP REES, T. (1959). Cytochrome oxidase in mycorrhizal and uninfected roots of Fagus sylvatica. New Phytol., 58, 364.

HARLEY, J. L. & MCCREADY, C. C. (1952). The uptake of phosphate by excised mycorrhizal roots of the beech. III. The effect of the fungal sheath on the availability of phosphate to the core. New Phytol., 5', 343.

HULME, A. C. & JONES, J. D. (I962). Tannin inhibition of plant mitochondria. In: Enzyme Chemistry of Phenolic Compounds (Ed. by J. B. Pridham). Pergamon Press, Oxford.

LEWIS, D. H. & HARLEY, J. L. (I965a). Carbohydrate physiology of mycorrhizal roots of beech. I. Identity of endogenous sugars and utilization of exogenous sugars. New Phytol., 64, 224.

LEWIS, D. H. & HARLEY, J. L. (I965b). Carbohydrate physiology of mycorrhizal roots of beech. II. Utiliza- tion of exogenous sugars by uninfected and mycorrhizal roots. New Phytol., 64, 238.

LEWIS, D. H. & HARLEY, J. L. (I965C). Carbohydrate physiology of mycorrhizal roots of beech. III. Movement of sugars between host and fungus. New Phytol., 64, 256.

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