CHANGES IN THE SIZE OF ORTHOPHOSPHATE POOLS IN MYCORRHIZAL ROOTS OF BEECH WITH REFERENCE TO ABSORPTION OF THE ION FROM THE EXTERNAL MEDIUM

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1 CHANGES IN THE SIZE OF ORTHOPHOSPHATE POOLS IN MYCORRHIZAL ROOTS OF BEECH WITH REFERENCE TO ABSORPTION OF THE ION FROM THE EXTERNAL MEDIUM BY D. H. JENNINGS The Botany Department, Leeds Universitv {Received 14 November 1963) SUMMARY At concentrations of potassium dihydrogen phosphate in the external medium below io"^ M, the greater proportion of phosphate which enters beech mycorrhizal roots is incorporated into bound form. Above io~3 j^i, an increasing proportion is accumulated unchanged as orthophosphate. Studies using S'^P confirm, in greater detail, the observation of Harley and Loughman (1963) that phosphate in the external medium rapidly equilibrates with a small pool of orthophosphate within the fungal sheath, from which the ion is incorporated into bound form or transferred either to the host core or to a larger pool in the sheath itself. Ammonium chloride at io~2 M increases the total uptake of phosphate through an increase in the rate of both the incorporation into bound form and the primary absorption process. Ammonium chloride over the range io-4 to io-^ M brings about an increase in the size of the small pool. However, ammonium chloride has no detectable eftect on phosphate fractions in the absence of phosphate in the external medium. Sodium azide, on the other hand, causes a breakdown of bound phosphate, although it is only at high concentration that this inhibitor has any detectable effect on the level of orthophosphate within the roots. The results suggest that the size of the small pool may be an important determinant of phosphate uptake at low concentration and that the role of potassium in the primary absorption process must not be overlooked. INTRODUCTION The data obtained with potato tuber slices (Loughman, i960; Bieleski and Laties, 1963) and beech mycorrhizal roots (Harley and Loughman, 1963) indicate that phosphate entering these tissues rapidly equilibrates with a small pool of orthophosphate which is closely associated with those metabolic reactions incorporating phosphate into bound form. This pool is about 5 % of the total orthophosphate in potato discs and for beech mycorrhizal roots contains about ^E ^^ phosphorus per 100 mg dry weight. In both cases, the size of the pool has been determined by indirect means and the estimates are subject to some degree of error. The present paper concerns the small orthophosphate pool in beech mycorrhizal roots and is specifically concerned with the size of the pool. It will be shown that the pool can vary considerably in size and it is suggested that such variations could be an important factor controlling the rate of phosphate uptake from the external medium. Some of these results have already been summarized and discussed in a much wider context (Jennings, 1963)- METHODS Beech mycorrhizal roots were collected from a stand of beech trees growing over boulder clay in Saw Wood, near Leeds. The root tips were prepared and sampled in the manner of Harley and McCready (1952a). Sample size varied from fifty to ninety root tips, i.e. D N.P. 181

2 i82 D. H. JENNINGS about mg dry weight of material. The excised root tips were left overnight in aerated, demineralized water at room temperature. The experimental treatment was as follows. Samples were shaken in ^^ of solution in stoppered 250 ml conical flasks. Hand shaking was used when short time intervals were involved, mechanical shaking was used for periods longer than 6 minutes. All solutions contained 5 io^^ ^ succinic acid and tartaric acid buffered to ph 5.5 with triethylamine. Potassium dihydrogen phosphate, labelled when appropriate with 32p (orthophosphate in dilute HCl from the Radiochemical Centre, Amersham), was used as the phosphate salt. The radioactivity varied between 2 and 10 pc/ml. Phosphate concentrations used in the experiments involving '^-P varied between 5 x io^^ ^ a^fj jo"^ M. It was considered that concentration of phosphate in this range was sufficiently low to provide a suitable specific activity yet sufficiently high to avoid an excessive proportion of the radioactivity being adsorbed on to the glassware. At the end of the regime in the required phosphate solution, the solution was decanted off and 200 ml of demineralized water at 2 C poured on to the roots. This marked the end of the experimental treatment. The roots were washed with three more aliquots of demineralized water at 2 C, transferred to filter paper, the surplus water rapidly removed, and the acid soluble phosphate extracted. 2 N HCl was used for extracting the acid soluble phosphates from the root tissue. Roots were ground with 5 ml of acid in a mortar containing a pinch of acid-washed sand, and the suspension of broken cells was centrifuged at 1500 g for 5-10 minutes, both operations at 2 C. Further extractions were made with 5 ml aliquots of a N HCl until a total of 25 ml had been added. The ph of the supernate was then raised to with 5 N ammonium hydroxide, the liquid being stirred continuously. The solution was then made up to 100 ml. The sediment was filtered off on to a Whatman No. 40 (7.0 cm) paper and then dried. The paper and sediment were boiled with 10 N H2SO4 in a 50 ml Kjeldahl Hask until all the material was made soluble. The boiling w^as continued for at least another 4 hours. After this period, the Hasks were allowed to cool, then 5 ml of 30 vol hydrogen peroxide were added. The liquid was boiled gently. When all the peroxide activity had ceased, the liquid was usually colourless. If this were not so, another 5 ml of peroxide were added and the procedure repeated. The clear solution was then made up to 50 ml. The analytical methods were as follows. Orthophosphate (P^) was determined by the method of Fiske and Subbarow (1925) modified by the presence of 10 % ethyl alcohol in the reaction mixture. For the determination of '7 minute hydrolysable phosphate' (P7), the quantity of orthophosphate in a known volume of solution was measured after 7 minutes m the presence of an aliquot of 2 N HCl at 100 C, followed by the addition of 10 N HCl to bring the solution to neutrality. The actual value obtained by this procedure IS the sum of both free orthophosphate and orthophosphate produced by hydrolysis. The total acid-soluble phosphate was determined as orthophosphate after hydrolysis w'lth H2SO4. Twenty millilitres of extract was heated with 4 ml of 10 N H0S04 until the liquid was reduced to small volume, when it might be either colourless or light yellow. A colourless solution was produced by further heating in the presence of 5 ml of 30 vol hydrogen peroxide. The quantity of orthophosphate in a known volume was determined after neutralization with 10 N NaOH. The amount of NaOH required was determined using a rephcate sample and methyl orange. The acid-insoluble phosphate (i.e. that in the sediment) w^as also determined as orthophosphate after digestion and neutralization.

3 Absorption of phosphate by beech mycorrhiza 183 Radioactivity was determined with an end-window Geiger-Miiller tube (Philips 18505). One millilitre of solution was used for all determinations and contained in a planchette of 20 mm diameter for counting. The activity ofthe solutions was unaffected by a concentration range of io^ M. The radioactivity in the orthophosphate fraction was determined by a method similar to that devised by Lindberg and Ernster (1956). This procedure, which depends upon the solubility of the phosphomolybdic complex in wo-butyl alcohol, is as follows: 0.4 ml of 10 N H2SO4, 0.6 ml of reducing agent (as used for the chemical determination of orthophosphate described above) and 0.8 ml of 2.5 % ammonium molybdate were added to a suitable quantity of extract in a graduated test tube, made up to 10 ml and shaken with 4 ml of wo-butyl alcohol. Three millihtres ofthe wo-butyl alcohol layer M'ere removed and shaken with 3 ml of deminerauzed water and 0.5 ml of 10 N H2SO4. One millilitre of the wo-butyl alcohol was removed for the determination of radioactivity. The same procedure was used for the determination of radioactivity in the 7 minute hydrolysable fraction after hydrolysis with 2 N HCl (and subsequent neutralization with 10 N NaOH). The actual value of radioactivity which is determined is the sum of both free orthophosphate and orthophosphate produced by hydrolysis. 5x10 Fig. I. The increase in phosphorus content of P, and P, within the roots as a result of exposure to a range of phosphate solutions for 12 hours at 23 C. There is no change in the phosphorus content of P,s and acid-insoluble phosphate. RESULTS Fig I shows the changes which occur in the various intracellular phosphate fractions as a result of absorption of phosphate ions for 12 hours from solutions of varying concentration between 2.5 X lo^^ M and IQ-^ M. Both P, and Pv increase in quantity. The latter shows the greater increase at low concentration of phosphate in the external medium, the increase in P, only occurring when the rate of incorporation into Pv tends to be independent of external concentration. There is no detectable change m the concentration of those compounds which are not hydrolysed by 2 N HCl within 7 minutes at 100 C

4 184 D. H. JENNINGS (P,s) nor in the concentration of acid-insoluble phosphate. A study of the metabolism of phosphate within roots following absorption from 5 X io~2 M solution over varying periods of time shows similar findings (Fig. 2), namely that at this concentration the major portion of the phosphate which is taken up by the roots remains as orthophosphate and that P7 is the only other fraction to increase in size. It appears that incorporation of phosphate into bound form plays an important role in the uptake of phosphate from low concentrations in the external medium. This role, however, diminishes in importance as the concentration is increased. Some indication of the importance of this incorporation process is given by the data presented in Fig. 3. Roots were pretreated with a range of phosphate solutions from io"^ M to io~^ M for 105 minutes in one experiment and 30 minutes in the other and the subsequent absorption eoo en o '- AOO o Hours Fig. 2. The increase in phosphorus content of P; and P7 within the roots at different intervals of time during exposure to 5 x 10-2 M phosphate solution at 22" C. There is no change in the phosphorus content of P5 and acid-insoluble phosphate. of radioactivity from 5 x io^s ^ phosphate over 5 minutes in the former experiment and over 8 minutes in the latter was determined. It can be seen that marked inhibition of absorption of radioactivity occurs when the concentration of the pretreating solution is more than 5 x io^s M. This indicates that, under conditions in which incorporation into bound form is likely to be reduced, the uptake of phosphate from low concentrations in the external medium is also reduced. Fig. 4 shows the distribution of radioactivity in the various phosphate fractions extracted from mycorrhizal roots of beech which had been exposed to 9 x lo-s M phosphate solution for 6 minutes. Radioactivity is incorporated into all the phosphate fractions within the root. The incorporation of 32p into P, and Pv is to be anticipated in view of the mcreases m these fractions which accompany phosphate absorption detected by chemical analysis. On the other hand, the incorporation of 32p into P, and acid-insoluble phosphate IS in contrast to the lack of any increase under the same conditions which could he detected by chemical analysis.

5 Absorption of phosphate hy beech mycorrhiza 185 For Vs, P7 and acid-soluble phosphate, there is a lag of about 2 minutes in the development of maxmum rate of incorporation which, when achieved, is constant over the remaining 4 minutes. The pattern of incorporation for these fractions can be ascribed to the dependence of each of these fractions on compounds within other fractions which act as precursors. On the other hand, for P^ the initial rate of incorporation is more rapid than the subsequent, constant rate which becomes evident after 2 minutes. This decline in rate of incorporation which occurs after 2 minutes is not due to the approach to an equilibrium between the total orthophosphate within the root and that in the external medium, for, as will be shown, incorporation of radioactivity continues at the slower rate for several hours. The simplest explanation of the curve of incorporation of radioactivity into Pj is loor 08O Si 60 Q " 10"^ Fig. 3. The effect of pretreatment in a range of phosphate solutions upon the incorporation of radioactivity from low concentrations of phosphate. A, pretreatment for 105 minutes and exposure to 5 x io"^ M phosphate containing 0.04 tic 32p/ml for 5 minutes at 2<f C. B, pretreatment for 30 minutes and exposure to 5 x io~'^ M phosphate containing ^"^ ^"P/ml for 8 minutes at 17 C. to assume that there are at least two pools of orthophosphate within the roots. One pool is rapidly labelled (possibly with almost maximum incorporation of radioactivity) in 4 minutes, the curve of incorporation approximating to a rectangular hyperbola. The other pool shows a very much slower rate of incorporation and, since maximum incorporation only occurs after several hours, the rate of incorporation is constant over the period examined. The curve for labelling of Pi is a summation of the curves of incorporation into the two pools. Exposure of roots to io^* M potassium dihydrogen phosphate for a much longer time period (Fig. 5) shows that the approximately linear increase in radioactivity in all fractions continues for at least 3 hours. Thereafter, whilst the radioactivity in the three other fractions continues to increase at the same rate, that in P7 shows little further increase with time, since equilibrium conditions with regard to the phosphorus within this fraction have been achieved. The data demonstrate what has already been indicated

6 186 D. H. JENNINGS 20 Minutes Fig. 4. The incorporation of radioactivity into the various phosphate fractions within the roots after varying periods of exposure to 9.1 >'. io"^ M phosphate containing 10.4 IJC ^^Pj/ml at 25 ' C. Columns indicate the phosphorus content of the fractions estimated by chemical methods. Fig. 5. The incorporation of radioactivity into the various fractions within the roots after varying periods of exposure to io-* M phosphate containing 2.0 uc 32p/ml at 22 C.

7 Absorption of phosphate by beech mycorrhiza 187 above, namely that the rate of incorporation of radioactivity into Pj which is achieved after the first few minutes remains constant for at least 7 hours. MetaboUc turnover of 32P or the initiation of eiflux of radioactivity cannot account for this linearity over such an extended period of time. Pretreatment of the roots with 10-2 M ammonium chloride for 12 hours leads to an increased rate of incorporation of radioactivity into all fractions. Data for 4 minutes are given (Fig. 6), but the increases can still be measured over a 4 hour period. Increased metabolic turnover of phosphorus is undoubtedly a cause of an increased rate of incorporation. A 50 % increase in the synthesis of P7 has been measured in the presence of 5 X 10^3 M ammonium chloride when roots are exposed to io~- M phosphate over a 12 hour period (there is no effect on the other fractions). Furthermore, Harley, McCready and Geddes (1954) have shown that 6 x io-3 M ammonium chloride can induce a large respiratory response in roots which have been stored for a suitable period in distilled water. -NH4CI 3 A 0 Minutes Fig. 6. The effect of the pretreatment of the roots for 12 hours at 22" C with io~- M ammonium chloride on the incorporation of radioactivity into roots after varying periods of exposure to 10^* M phosphate containing 2.7 MC ^^P/ml at 23' C. Incorporation of radioactivity into Pi demands the most detailed examination. It has been postulated that the first phase of the Pj curve represents the labelling of the smaller of at least two orthophosphate pools. If this is so, the initial slope of the first phase, that is the rate at which the pool becomes radioactive, will be determined by the ratio of the rates at which radioactivity enters into and is lost from the pool and the size of the pool. The increase in the initial slope produced by the ammonium chloride pretreatment could be due to an accelerated rate of entry, a reduction in the rate of loss or in an increase in the size of the pool. There is every indication that the rate of loss is in fact increased, so that ammonium chloride must induce either an increase in the rate of entry of radioactivity into the pool or an increase in size of the pool itself both increases, indeed, are likely to occur. On the other hand, the point of inflexion of the curve, namely the total quantity of radioactivity in the pool when equilibrium is achieved will depend upon the

8 188 D. H. JENNINGS size of the pool and the turnover of orthophosphate between the pool and other fractions. The increased level of radioactivity in the smaller orthophosphate pool induced by ammonium chloride could be caused either by an increased concentration of orthophosphate or a reduction in the rate of metabohc turnover. The undoubted increase in metabolic turnover at a stage when it is likely that atoms which are predominantly unlabelled are being fed back into the pool must mean that the increased level of radioactivity in the orthophosphate pool can only indicate that there has been an actual increase in the orthophosphate concentration within the pool. > Minutes Fig. 7. The redistribution of radioactivity between the various phosphate fractions within roots during a regime in phosphate-free buffer at 23 C. The roots were pretreated for 5 minutes in 4.2 x io-5 M phosphate containing 8.3 v-c ^^Pjml at 25 C. Transference of roots pretreated for 5 minutes with radioactive phosphate at 5 X io M to a phosphate-free medium results in a redistribution of radioactivity for which Fig. 7 gives characteristic data. The results from four experiments indicate that the main changes are: (i) A rapid fall in the radioactivity in Pj (nearly 50 % of the radioactivity is lost from this fraction in 5 minutes) followed by a slow rise. (ii) A fall in the level of radioactivity in Pj. This fall is less rapid than that for Pj and indeed there may be an initial rise but this has not been consistently observed. This fall is followed by a slow rise in the level of radioactivity. (iii) A rise in the level of radioactivity in P7 and acid-insoluble phosphate. The radioactivity in P7 is subsequently lost, that in the acid-insoluble phosphate is retained. It is not possible to discuss all the changes which are observed; nevertheless the data does provide valuable information about the orthophosphate in the root which is the primary interest in this investigation. The fact that radioactivity is lost so rapidly from Pi IS striking confirmation that the roots are not homogeneous with respect to orthophosphate and that there exists a small pool which equilibrates very rapidly with respect to the radioactivity which has entered the root. A smgle pool of orthophosphate of the size mdicated by chemical determinations would not lose radioactivity at the rate which IS observed unless an inordinately high rate of metabohc turnover were to be postulated. The subsequent rise in the level of orthophosphate may be due to either transference of radioactivity into a larger pool or the return of radioactivity into the smaller pool as the result of metabohc turnover, the involvement of both pools in this manner is not mutually incompatible. 50

9 Absorption of phosphate by beech mycorrhiza 189 The presence of ammonium chloride over a wide range of concentration (io^^io-i M) markedly reduces the loss of radioactivity from P* which occurs when the roots are transferred to a phosphate-free medium (Table i). Since there is no indication that ammonium chloride increases the rate of entry of radioactivity into Pj from another fraction, this decreased loss from Pj can only be due to an increase in size of the small pool of orthophosphate. Examination of changes over an extended period shows that over a period of 2 hours the level of radioactivity in Pj remains for the most part consistently higher in the presence of io^i M ammonium chloride, although it should be pointed out that reduction in the rate of metabolism may be responsible for these higher Table i. The effect of ammonium chloride on the radioactivity in Pi in roots stored for varying intervals of time in a phosphate-free solution after pretreatment for 5 minutes at 25^" C in a radioactive phosphate solution Results given in io^ counts/min per ioo mg dry wt. Time in solution (minutes) Concentration of ammonium chloride (molar) Initial level of radioactivity Level of radioactivity after storage Solution -NHjCl +NH4CI (a) Pretreatment in 5 ' o 5 phosphate, 10 w ^'-^ io~ 10^ storage at 25^ C (b) Pretreatment in 4.2.:io"5 M phosphate, 8.3 Mc '^'^Pjml, storage at 22 C o lo-i (c) Pretreatment in 4.2 / io"-' M phosphate, 8.3 v-c '-^-Plnil, storage at 23 ' C o io-i B levels towards the end of the experimental period. An extended period in 10 ^ M ammonium chloride can certainly markedly affect the physiological properties of the roots (Jennings, 1964). Thus, ammonium chloride can affect markedly the pattern of metabolism of orthophosphate entering the roots from the external medium. On the other hand, ammonium chloride has no effect on the chemical levels of the phosphate fractions within the roots when they are in a phosphate-free medium. It is particularly interesting that this should he true for Pj, even when the level of this fraction has been increased very considerably by the previous exposure of the roots to 10-2 M phosphate for 17 hours. In contrast, sodium azide causes the breakdown of all fractions within the root with accompanying

10 190 D.H. JENNINGS loss of orthophosphate into the external medium (Fig. 8). At the lower concentrations of azide, most of the orthophosphate which appears in the medium comes from breakdown ofthe combined phosphorus, very little being lost from the orthophosphate fraction itself. It is only above ro^^ M azide and at concentrations which also cause marked inhibition (as opposed to a stimulation at lower concentrations) in oxygen uptake and carbon dioxide production (Harley and McCready, 1953; Jennings, 1958) that orthophosphate is lost directly from the fraction in the root. It should be noted that phosphate uptake is inhibited over the whole range of azide concentrations used in the above experiment, with maximum inhibition at the highest azide concentration (Harley et al., 1956). M NaNj Fig 8. The effect of varying concentrations of sodium azide on the various phosphate tractions within the roots. Duration of experiment, 12 hours, 25" C. DISCUSSION The present observations confirm those of Harley and Loughman (1963) with respect to the pattern of mcorporation of radioactivity from low external concentrations of orthophosphate and the redistribution of the radioactivity when the roots are returned to a phosphate-free medium. It has been shown in this paper that not only the rate of loss of radioactivity from P, but also the initial rate of labelling of this fraction are commensurate with the presence of a small pool of orthophosphate in the roots. Phosphate ions entering the tissue equilibrate rapidly with this pool and it is from this pool that the ion is incorporated into combined form and also transferred to other locations inside the root. At low concentrations of phosphate only 10 % of the radioactivity which is absorbed is transferred to the host core (Harley and McCready, 1952^,). It is now known (Harley and Loughman, 1963) that 80 % of the radioactivity which is transferred is incorporated into

11 Absorption of phosphate by beech mycorrhiza 191 the orthophosphate of the host tissue. It is, therefore, possible to calculate the incorporation of radioactivity into P,; of the core and by difference into P^ of the fungal sheath. Thus, there is good reason for postulating (Fig. 9) that there are two larger pools of orthophosphate within the tissue, one in each component. The data also indicate that the rates of transfer of radioactivity from the small pool of orthophosphate in the fungal sheath to the larger pool in the same tissue and to the host tissue may be little different. If this is so, there would be good reason for ascribing the slow rise in radioactivity of P/, which is observed after about 20 minutes when the roots are placed in a phosphate-free medium, both to transference to the larger pool of orthophosphate in the fungal sheath and to the temperature-sensitive movement of radioactivity to the host core observed by Harley and Brierley (1954). Fig. 9. The incorporation of radioactivity into the orthophosphate of the fungal sheath and host core calculated from the data given in Fig. 5. At low external concentrations, the rate at which phosphate enters the root is governed by the rate of the primary absorption process. At these concentrations, phosphate is removed from the small pool (primarily by incorporation into bound form) more rapidly than it enters. This would account for the linear rate of uptake over an extended period from concentrations less than 3x10-4 j^i (Harley, Brierley and McCready, 1954). Above io~3 M, the rate of uptake is determined by the rate at which orthophosphate is accumulated in the root, both in the fungal sheath and the host core, for it is above this concentration that an increase of Pj can be demonstrated. This interpretation is supported by the fact that pretreatment of roots for relatively short periods of time with concentrations of phosphate greater than 10 ^ M very markedly reduces the subsequent uptake from 5 x io-^ M phosphate, whereas concentrations less than io^^ ^ have little effect. The present observations also show that the small pool of orthophosphate in the fungal sheath can vary in size. At this stage, it is not appropriate to give an explanation of the increase in size of this pool. However, it is clear that any increase in size of the pool ought

12 192 D. H. JENNINGS to be reflected in the initial rate of transfer of radioactivity to the host. An analysis of the movement of radioactivity across the fungal sheath similar to that made by Harley et al (1954) ought to confirm by a quantitative estimate the increase in size of the orthophosphate pool. There is as yet little indication as to how changes in the concentration of the small pool of orthophosphate may affect the entry of the ion from the external medium. If the primary absorption process is dependent upon a difference in concentration, the rate of uptake from low concentrations of phosphate could be reduced by an increase in the size of the pool. The decrease in uptake from 5 x io"5 M phosphate brought about by pretreatment with concentrations of io"^ M phosphate might be ascribed, in part, to the increase in pool size. While it has proved difficult to devise a convincing experimental test of this hypothesis, it is pertinent to point out that this sort of consideration is relevant to the observations on the rate of uptake by fresh and aged potato discs. Loughman (i960) and Bieleski and Laties (1963) have shown that the storage of potato discs for 2 days in aerated distilled water increases the rate of uptake from io-^ M phosphate by more than 100-fold but has comparatively httle effect on the rate of uptake from 10-2 M phosphate. A decrease in the cytoplasmic, orthophosphate pool brought about hy the increased esterification which is observed during the ageing process could be a primary cause of the above difference. It has been pointed out that at low concentrations the rate of uptake of phosphate is determined by the primary absorption process. It is therefore clear, if the above argument is correct, that, since ammonium chloride can bring about both an increase in pool size and the rate of phosphate uptake from low concentration in the external medium, the rate of the primary absorption process is also increased under these conditions. An increased synthesis of an hypothetical carrier could account for the change in rate, but a greater uptake of potassium in consequence of the increased metabolism, in the manner suggested by Harley and Wilson (1959), could be equally effective. The results with azide give an indication of the possible importance of potassium in the absorption of phosphate. Harley and Wilson have shown that this inhibitor, at the same concentration range as above, brings about large losses of potassium from mycorrhizal roots of beech. At concentrations of azide below io-s M, phosphate could be lost as the accompanying anion m consequence of the increase in size of the small pool, due to the release of phosphate from bound form. Whatever the mechanism of loss of phosphate under the influence of azide, the results obtamed with this inhibitor at low concentration and the lack of effect of ammonium chloride upon the level of P, clearly show that the major portion of orthophosphate within the root IS in an inaccessible location and hence metabolically inactive Only those high concentrations which seriously inhibit the metabolism and possibly affect the structural integrity of the cells bring about any marked mobility of this major portion of orthophosphate. A final poi^qt concerns the metabolism of phosphate in bound form. Chemical analysis has shown that when phosphate enters the root an increase in phosphorus content can only be detected in P, and P,. No increase can be detected in P, and acid-insoluble phosphate, even when the roots have been exposed to 10-2 M phosphate for 23 hours. Radioactivity data show that these latter two fractions are certainly metabolically active. It must be presumed, therefore, that the eventual equilibrium in excised roots in a naedium containing phosphate is very much in favour of the transference of phosphate to the larger pool of orthophosphate m the fungal sheath and to the host core, and of the net

13 Absorption of phosphate by beech mycorrhiza 193 synthesis of compounds in P7. This equilibrium develops to the exchision of detectable net synthesis of compounds in P^ and the acid-insoluble phosphate fraction. REFERENCES BlELESKI, R. L. & LATIES, G. G. (1963). Turnover rates of phosphate esters in Fresh and ascd slices of potato tuber tissue. Plant PhysioL, 38, 586. FiSKE, C. H. & SUBBAROW, U. (1925). The colorimetric determination of phosphorus, jf. biul. Chen/., 61, 375- HARLEY, J. L. & BRIERLEY, J. K. (1954). The uptake of phosphate by excised mycorrhizal roots of the beech. VI. Active transport of phosphorus from fungal sheath to host tissue. New Plivtol., 53, 240. HARLEY, J. L., BRIERLEY, J. K. & MCCREADY, C. C. (1954). The uptake of phosphate by excisi;d mycorrhizal roots of the beech. V. The examination of possible sources of misinterpretation of the quantities of phosphorus passing into the host. Nezv Plivtol., 53, 92. HARLEY, J. L. & LOUGHMAN, B. C. (1963). The uptake of phosphate by excised mycorrhizal roots of the beech. IX. The nature of the phosphate compounds passing into the bost. New Phytol., 62, 350. HARLEY, J. L. & MCCREADY, C. C. (1952a). Tbe uptake of pbosphate by excised mycorrbizal roots of tbe beech. II. Distribution of phospborus between host and fungus. N'ew Phytol., 51, 56. HARLEY, J. L. & MCCREADY, C. C. (19526). Tbe uptake of pbosphate by excised mycorrbizal roots of tbe beech. III. Tbe effects of tbe fungal sbeatb on tbe availability of phospbate to tbe core. New Plivtol., HARLEY, J. L. & MCCRE.ADY, C. C. (1953). A note on tbe effect of sodium azide upon tbe respiration of beech mycorrbizas. Netv Phytol., 52, 83. H.-\RLEY, J. L., MCCREADY, C. C, BRIERLEY,'J. K. & JENNINGS, D. H. (1956). Tbe salt respiration of excised beecb mycorrbizas. II. Tbe relationship between oxygen consumption and phospbate absorption. New Phytol., 55, i. H.^RLEY, J. L., MCCREADY, C. C. & GEDDES, J. A. (1954). Tbe salt respiration of excised beecb mycorrbizas. I. Tbe development of tbe respiratory response to salts. Netv Phytol., 53, 429. HARLEY, J. L., & WiLSON, J. M. (1959). Tbe absorption of potassium by beecb mycorrbiza. New Plivtol., 58,281. JENNINGS, D. H. (1958). Cbanges in tbe internal carbobydrates of beecb mycorrbizas during treatment witb azide. New Phytol., 57, 254. JENNINGS, D. H. (1963).' The Absorption of Solutes by Plant Cells. Oliver & Boyd, Edinburgb. JENNINGS, D. H. (1964). Tbe effect of cations on tbe absorption of pbospbate by beecb mycorrbizal roots. (In preparation). LiNDBERG, O. & ERNSTER, L. (1956). Determination of organic pbospborus compounds by pbospbate analysis, Meth. hiochem. Anal., 3, i. LOUGHMAN, B. C. (i960). Uptake and utilization of pbospbate associated witb respiratory cbanges in potato tuber slices. Plant PhysioL, 35, 418.

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