THE UPTAKE OE PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OE THE BEECH

Transcription

1 [ 24O ] THE UPTAKE OE PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OE THE BEECH VI. ACTIVE TRANSPORT OF PHOSPHORUS FROM THE FUNGAL SHEATH INTO THE HOST TISSUE BY J. L. HARLEY AND J. K. BRIERLEY Department of Botany, University of Oxford {Received 23 June 1953) (With 3 figures in the text) There are two kinds of route by which phosphorus might pass from the external solution into the host tissue of beech mycorrhizas. Movement might be by way of the interhyphal spaces or the walls of the fungal tissue, or through the living hyphae themselves. Although several types of factor such as oxygen concentration and phosphate concentration in the external solution have been shown to alter the relative rates of absorption of phosphorus by sheath and core, no unequivocal experimental evidence has been presented which demonstrates which of these routes is taken in any given experimental conditions (Harley & McCready, 19526). The present paper describes experiments which provide information on this problem. In these, excised mycorrhizal roots were allowed to absorb radioactive phosphorus during the first phase of each experiment. In the second phase the distribution of the absorbed radioactive phosphorus in the fungal and host tissue was determined after known time periods in phosphate-free buffer solution under conditions in which the rate of metabolism was varied. METHODS The plant material was sampled and prepared as in all previous work. Within i or 2 hr. after collection the roots were placed in a solution of potassium phosphate labelled with ^-P. This solution was called the charging solution. In all experiments it consisted of o-i6i mm phosphate (5 mg. P per litre) buffered at ph 5-5 in o-oi M phthalate and in it the roots were agitated at 25'^ C. in a thermostat for 1-4 hr. After charging the roots were rinsed and placed in fresh phosphate-free buffer (ph 5-5) in aeration tubes (Harley & McCready, 1952a), through which bubbles of any desired gas mixture were passed. This solution was called the washing solution. During the washing period the aeration tubes were kept at a constant temperature in an incubator or refrigerator. At the end of the charging period one set of samples was dried immediately and further samples of six to twelve roots were dissected. Erom these two sets of samples the total phosphate absorbed and the distribution of '^-P between fungal sheath and host core could be estimated. The remaining samples were washed under controlled conditions before being dried or dissected for estimates of total residual radioactivity and its distribution between fungus and host.

2 uptake of phosphate by excised mycorrhizal roots 241 Since the movement of radioactivity from sheath to core during the washing period was found to be temperature sensitive the method of handling the charged roots was slightly modified as compared with previous work. All the rinsing of samples was carried out with ice-cold water, and roots awaiting dissection were kept in a refrigerator. The material obtained by dissection of each root was separately Geiger-counted to estimate the relative radioactivity of its component sheath and core tissues. The data obtained by dissection were, on account of the additional precautions taken, less variable than in previous work. The estimates of variability of these samples cannot be applied to any previous results obtained by other techniques. Each experiment involved two separate series of estimatesthe estimate of total ^^P in the samples on a dry-weight basis and the estimate of the proportion of ^^P in the cores of the roots obtained by Geiger-counting dissected samples whose weight was unknown. The mean values of these two sets of measurements were used to obtain an estimate of the amount of ^^P in the cores of 100 mg. of dry roots. The variance of the quantity of ^^P in the core was estimated by the function p\ Vw) + w\ Vp), where w is the mean total ^"P per 100 mg. of dry roots and p is the mean proportion of ^^P in the cores of the samples dissected and [Vw) and (Vp) their respective variances. The contribution of the term p^{vw) to the total variance was in all cases small compared with the contribution of w^{vp). In the experiments shown in Tables 3, 6 and 8, and Figs, i and 2, it was inexpedient to estimate enough total ^^P values to calculate (Vw). The comparisons therefore make use only of the variance of the dissected material and are not quite so reliable as those of other experiments. METHOD OF PRESENTATION OF THE RESULTS It has been shown (Harley, Brierley & McCready, 1954) that during an absorption period the total amounts of phosphorus absorbed by beech mycorrhizas and the amounts of phosphorus retained in sheath and core can be calculated satisfactorily from estimates of radioactivity. This is because the specific activity of the mobile phosphate fraction in the sheath is not significantly different from that of the external solution after a short period of absorption from solutions of very low concentrations of phosphate. It cannot be assumed, however, that this is true during a period of washing for there might be a conversion of phosphate from a non-mobile to a mobile form within the sheath during washing. For this reason, in the tables of experimental results given below, the quantities of radioactive phosphorus moving from sheath to core during washing periods are given on an arbitrary scale. The actual units of the scale are Geiger counts per unit time per unit quantity of tissue divided by the specific activity of the charging solution that is Geiger counts per /xg. of phosphorus in the charging solution. If the specific activity of the mobile phosphorus in the sheath throughout the washing period could be assumed to be equal to that of the charging solution the arbitrary units would be /ixg. of phosphorus. This assumption cannot yet be made with confidence, and here the values given will be treated as minimum estimates of the amount of phosphorus moving from the sheath to the core. The same considerations apply to experiments which show a loss of total phosphorus from the roots during washing. The amounts recorded as lost will also be treated as minimum estimates of these losses.

3 242 J. L. HARLEY AND J. K. BRIERLEY EXPERIMENTAL RESULTS In the experiment summarized by Fig. i, mycorrhizal roots were charged with labelled phosphate for 75 min. in a buffer solution containing 5 mg. P per litre. Graph A shows the quantity of ^T present in the cores of roots immediately after charging and at timed intervals during the washing period up to 90 hr. At each point the mean quantity of ^T in the cores and the standard error of the dissections is given. The rate of movement of Time (hr.] Fig. I. A: quantity of '"P in arbitrary units per ioo mg. dry roots in the core tissue of mycorrhizal roots immediately after absorption of phosphate and at intervals during subsequent washing in phosphatefree buffer ph 5'5 at room temperature. Standard errors of estimates made by dissection are plotted for each point. B: total ^-P per ioo mg. dry roots after various periods of washing. Expt '^P from the sheath to the core is rapid at first, and after 15 hr. of washing the rate of movement decreases. Graph B in Fig. i shows that during the whole period of washing no loss of total ^"P from the roots occurred, a result which agrees with those of previous experiments (Harley & McCready, 1952a; Harley, McCready & Geddes, 1954) and with those of other experiments described below (Tables 4, 5, 7 and 8). The shape of graph A, Fig. i, suggests perhaps that the process of movement of ^^P from sheath to core may tend to exhaust a mobile phosphorus fraction in the sheath. This possibility is not considered further in this paper. The aim of these experiments is to determine whether the net rate of movement of *^P from sheath to core during periods of 15 hr. or more is affected by external conditions operating during washing. For this purpose the conditions of charging and subsequent treatment have been standardized as far as it has been expedient.

4 uptake of phosphate by excised mycorrhizal roots 243 Two kinds of experiment were performed to determine whether metabolic processes in the sheath and core were involved in the movement of ^^p which occurs during washing in phosphate-free buffer. The first set of experiments examined the effect of the temperature of the washing solution upon the rate of movement. The second set estimated the rate of movement during washing in solutions agitated with gas mixtures containing various oxygen concentrations. The effect of temperature on the rate of movement The results of four experiments upon the effect of the temperature during washing are shown in Table i. In the first two, washing at 3 C. is compared with washing at 19 C. In the second two, a lower temperature 1 C, was used instead of 3 C. In every case, a period of washing of 17^-19^ hr. at 19 C. resulted in a movement of 3-P into the core from the sheath so that the quantity in the core might even double during that time. At 3 C. movement was very slow, and only in one of the experiments was a statistically significant movement recorded. At 1 C. movement was negligible in both experiments. Table i. The results of four experiments showing the effect of temperature upon active transport of ^^P from sheath to core Charging times ij, 2, 2J and 2^ hr. respectively. Washing times 17J, 19, 19, 19^ hr. respectively. Results calculated by pooling total ^^P readings for all treatments to ohtain estimate of variance. Variance of ^^P in cores obtained hy zii\vp)-\-p''(vw) vi'here zt' = mean total ^^P in the roots and ^ = mean proportion of '^P in cores. Significance of results tested by E test. Estimates of ^''P in arhitrary units (see text). : Expt. no Treatment 19 C. 3 C. 19 C. 19 C. i C. 19=0. "P in I 00 mg. dr\' r oots Estimates i i8-4 i8-3 i I5'6 j I p / ' I 1-8 f [ 2-7 \ 1-7 I ^ I 3-3 ) " n core Difference + O O Difference for significance p_q.q_ O O'4O P = O'OOI 0-71 ) [ P = o-ooi j O-88 ) 0-83

5 244 J- L- HARLEY AND J. K. BRIERLEY A similar result is shown in Fig. 2, where the means and standard errors of the dissections are plotted for a fifth temperature experiment to compare the progress of movement at high and low temperatures. The progress of movement is again seen to be divisible into two phases at 19 C. These five experiments all indicate that the movement of ^^P from the sheath to the core is temperature-sensitive. Indeed, the temperature coefficient of each experiment is higher than 2 and the results are in agreement with the suggestion that the movement is dependent on metabolic processes Time (hr.) Fig. 2. Quantity of ^^P in arbitrary units per 100 mg. dry roots in the core tissue of mycorrhizal roots immediately after absorption of phosphate and at intervals during washing. A, washed at 19 C; B, washed at i C. Standard errors as Fig. i. C shows the total '^P per ioo mg. dry roots after various periods of washing. O, washed at 19 C; + washed at 1 C. Expt The results of the two experiments shown in Table 2 demonstrate that after a period of washing at low temperature, during which transport of radioactive phosphorus from sheath to core is slow, the movement may increase in rate on return to a higher temperature. The reduction of the rate of transport in the cold is not therefore due to a mobile fraction of phosphorus being utilized in the sheath but is, at least in part, due to a cessation of the transporting reactions at low temperature. The effect of oxygen concentration upon the rate of movement Table 3 gives the results of two experiments in which series of samples were washed in buffer agitated with air or with nitrogen. The buffer solutions were treated in the manner previously used (Harley, McCready & Brierley, 1953) to bring them into equilibrium with the appropriate gas, and they were then agitated with bubbles for 8-i hr. before the charged roots were introduced.

6 uptake of phosphate by excised mycorrhizal roots 245 Table 2. Results of two experiments in which charged roots were kept at first at low temperature and transferred to a higher temperature later values are given of the radioactivity in the cores of roots after charging and after the washing treatment, in arbitrary units. Expt. no. Treatment ist phase '^P in core Treatment 2nd phase ^*P in core i9jhr., 1 C. I9i hr., 19" C hr., 19 C hr., i C. 21 hr., I'C. 27 hr., i' C. 42 hr., i' C '4 18 hr., 18 C. 10 hr., 18 C. IS hr., i8 C O Table 3. Comparison of the rates of active transport of ^^P from sheath to core of roots kept in washing solutions agitated with air or Ng at room temperature Charging times 4i and 3 hr. Washing times 17^ and i6i hr. Amounts in the core calculated from ons using the mean value for total' ^Pin roots of the same treatment. Expt. no. 32p per 100 mg. dry roots Estimates 32p n core Difference 8. IO II. 52 Air Difference between core ( j means for significance ) Air Difference between core means for significance i> = oo O 2-9 P = o-o O o-o O-57 Two important points are illustrated by these results. First, the roots kept in nitrogen during the washing period appeared to lose phosphorus since the mean total radioactivity per unit weight fell. Secondly, the movement of radioactive material from sheath to core in nitrogen, as calculated from the total radioactivity of the samples kept in nitrogen, was much less than in air. Neither in air nor in nitrogen did ^^p in the core diminish, the loss of radioactivity either to the core or to the external solution was from the sheath. Since the loss of *T into the washing solution in nitrogen seemed to introduce a complication, the experiment shown in Table 4 was performed. Eight replicate samples New Phytol. 53, 2 i5

7 246 J. L. HARLEY AND J. K. BRIERLEY for total ^^P in the control and in each treatment were used to obtain a statistical apprisal of the apparent losses during washing in nitrogen. The losses were shown to be significant to a probability of a thousand to one. The replication of estimates of total ^^P absorbed and of dissections also allowed a more reliable assessment to be made of the relative rate of movement of ^^P from sheath to core in air and in nitrogen. The movement in air was shown to be highly significant, whereas in nitrogen no significant movement of ^^F from sheath to core occurred. Table 4. Residts of Expt which show that ^^P is lost from roots washed in anaerobic conditions and that there is insignificant active transport into the core as compared with aerobic conditions Charging time 3i hr. Washing time 19!^ hr. quantity of ^''P in core samples and their variances calculated from total samples kept in identical conditions. s of total '^Pfrom eight samples, dissections from ten samples in each treatment. Treatment Total '^P per 100 mg. dry roots Diff. Diff. for significance ^^P in core Diff. Diff. for significance Air o-s -2-3 \ P=O'OOI I /> = oos ' O-3 j P = oooi 1 O-74 P = o-o5 O'4i These two sets of experiments, comparing movement at low temperatures and high temperatures and in aerobic and anaerobic conditions, show that movement of ^^P into the core depends in rate upon temperature sensitive and oxygen sensitive processes in mycorrhizal roots. Three simple but incompatible hypotheses concerning the mechanism of movement are the following. (1) The phosphorus which moves into the core during washing is solely part of that which is present in the interhyphal spaces of the sheath at the end of the charging period, having arrived there by diffusion from the charging solution. (2) A leakage of phosphorus from the sheath tissue into the interhyphal spaces is constantly occurring during washing. In conditions where the metabolic rate of the core is rapid, part of the phosphorus in these spaces is rapidly absorbed into the core. Hence at normal temperatures in air a movement of ^^P from sheath to core occurs, but in nitrogen ^-P is lost to the washing solution. (3) The movement of ^-P from sheath to core depends upon metabolic activity in the sheath and core so that significant quantities move only in air at normal temperatures. The loss of ^T from the sheath in nitrogen would be interpreted in this case as a secondary effect having no bearing on transport. The first of these hypotheses is shown to be improbable from a consideration of the quantities of labelled phosphate involved. Where a movement into the core of two arbitrary units of ^^P per ioo mg. of dry roots occurs during washing this is equivalent to the 32p contained in about 0-4 ml. of charging solution. The total fresh volume of 100 mg. of dry roots is only about 07 ml. (Harley et al. 1953), and of this only about onequarter is occupied by the sheath. Hence the whole of the mobile phosphorus could not be contained in the whole volume of the sheath unless it were accumulated there to a higher concentration than that of the charging solution.

8 uptake of phosphate by excised mycorrhizal roots 247 If the second hypothesis were true it might be expected that there would be a similar loss of ^^P from the roots in all conditions where the rate of accumulation in the core was greatly reduced. It may be seen from Tables i and 5 that there is no evidence of loss of *^P from roots kept in the cold. This contrasts sharply with magnitude of the loss from roots kept in nitrogen (Tables 3 and 4). Hence from the foregoing experiments the balance of evidence is against the first two hypotheses. Table 5. Comparison of samples washed at high and low temperatures in air showing that the loss of ^^P from mycorrhizal roots is negligibly small in these conditions Charging time 3 hr. Washing time 14^ hr. Five samples in each treatment. Expt Total ^'P per 100 mg. dry roots Difference from control C C O-2 Difference for P=o-O In a previous paper upon the effect of oxygen concentrations upon the uptake of '^P by sheath and core it has been shown that whereas the sheath is very sensitive to oxygen the core is much less so (Harley et al. 1953). Indeed, experiments with intact roots and separated cores showed that the rate of phosphorus uptake by the core was almost unaifected by reduction of the oxygen concentration from 21 % to about i %. These previous observations may be used to distinguish experimentally between the above hypotheses. In the experiments shown in Table 6 the movement of ^T into the cores of roots during washing in air, nitrogen and in a series of low oxygen concentrations is compared. It would be expected that if the first hypothesis were true the rate of movement into the core would be almost unaffected down to about i % oxygen. Secondly, if the second hypothesis were true the rate of movement into the core would either be constant over the same range or would increase at low oxygen concentrations. Neither of these conditions is fulfilled. Table 6. Results offour experiments comparing rate of active transport of phosphorus from sheath to core in roots kept in phosphate-free buffer ph 5-5 agitated with various gas mixtures Charging times 4, 3, 3 and 2J hr. Washing times 18J, I9i, i6f and 15! hr. respectively. Amounts of '^P in cores calculated from the mean of estimates of total '^P for that treatment. xpt. no.... inwashed) 21% 5-6% 3-o% 1-5% 0-7% Difference P=o-O between Total '''P per ioo mg. dry 1-oots mean for significance Diff Diff ' 'i II. 52 Diff O-I O-I Diff i-i + i-i + I-I

9 248 J. L. HARLEY AND J. K. BRIERLEY The experiment in Table 7 examines these points in greater detail using air, 3 % oxygen and pure nitrogen. The concentration of 3 % oxygen was chosen for detailed study because it is well above the level at which core uptake is significantly affected by oxygen concentration and is at the same time close to the 50 % value for phosphorus uptake by the sheath (Harley et al. 1953). The replication of samples enables the conclusion to be drawn that at 3 % oxygen a significant loss of phosphorus into the washing solution occurred,* but there was no significant gain of ^'-P by the core. The available evidence based on many separate experiments on the effect of oxygen concentration on uptake of phosphate by host and fungus and upon the active transport of ^^P from sheath to core is summarized in the graphs shown in Fig. 3. In each case the radioactivity of the plant material in the various oxygen concentrations is plotted as a percentage of the radioactivity in air. It may be seen that the effect of oxygen concentration upon movement of ^-P from sheath to core closely resembles that upon uptake by the fungus, and differs strikingly from that upon uptake by the host. It is reasonable, therefore, to assume that transport of ^"P from fungus to host during washing is dependent upon oxidative reactions which occur in the fungal tissues and perhaps in a lesser degree only upon those of the host. In conditions where the metabolic activity of the sheath is reduced by low temperatures or by low oxygen concentration transport of phosphorus into the core during washing is diminished. The loss of phosphate from the sheath into the external solution in low oxygen concentrations is secondary and may be associated with anaerobic processes occurring in that tissue. Table 7. Results of Expt which show that '-P is lost from roots washed in buffer agitated with Nj and 3% O2 but not from those washed in air. No significant active transport occurred in Ng or 3% O2 Charging time 3 hr. Washing time 17 hr. The mean quantities of '^P in the cores and their variances calculated from mean quantities of total ^^P in samples kept in appropriate conditions. of total '''P of five samples, dissections of eight samples. Total ^^P per 100 mg. dry roots '^P in core Treatment DifT. from control Diff. for significance -^ 1 Diff. from '^^''", control DifT. for significance Air 3 % 0, N, O-3 i"4-2-3 ^ P=o-ooi 1 2'OI 1 P=o-oi ( 1-46 P=oo5 j I-06 3'5 5-7 ' O ' O ' P = o-ooi 206 P = OOI 1-55 P = o-o5 In Table 8 the results of an experiment are given which compare the loss of ^^P in anaerobic washing solution at high and low temperatures. The loss is much reduced at a temperature of 4-5 C. as compared with 23 C. so that the 0^ of the process of loss is high (2-3). It appears possible therefore that the release of phosphate into the external solution in nitrogen depends in rate on a chemical reaction which proceeds anaerobically and is not due to a simple cessation of the aerobic accumulatory processes. Since losses of phosphorus from the sheath tissue occur in anaerobic conditions it is of importance to determine whether after a period of washing in nitrogen, active transport Qf 32p from sheath to core is possible when roots are returned to aerated washing solutions. * In all subsequent experiments with 3% oxygen no loss of'^p has been observed, although in every case there was no significant gain of ^^P by the core.

10 uptake of phosphate by excised mycorrhizal roots c c - o o/ 8/ o I 0 o o 0 0 _ o 6 e IS 20 t_ fd c of absor-ption II centage Per 100 _ 75 " o / ^ SO - /o / -I 25 I 9" 1 ^ o ^^^'' c /b 50 "b o 25 / o Q - /o IS 20 o 0 Fig. 3. Comparison of the effect of oxygen concentrations upon the uptake of phosphate from o-i6imm potassium phosphate by the sheath and core of intact mycorrhizas and of fungal and host tissues with their effect upon active transport. In each case the uptake and active transport in air is plotted as 100 %. A: phosphate uptake by the sheath; B: phosphate uptake by the core; C: phosphate uptake by the fungal tissue; D: phosphate uptake by the host tissue; E: active transport of ^^P from sheath to core. Graphs A and B are based upon three experiments each ( , , ). Graphs C and D are based upon two experiments each ( , ). Graph E is based upon ten experiments, a sample of which is given in Table 6. In each experiment used to construct the graphs, a control in air was always included. The graphs are drawn hy eye.

11 250 J. L. HARLEY AND J. K. BRIERLEY Table 8. Comparison of the quantities of ^^P in arbitrary units lost from roots kept in aerobic conditions at 24 C. and in anaerobic conditions at 24 and 4-5 C. Charging time 3J hr. Washing time 23^ hr. Expt Air washed 24 C. Nj washed 24" C. No washed 4'5' C. Total ^^P per 100 mg. dry roots '''P lost - i-o 4-8 I-o For significance P = o-o5 difference between means must be greater than Table 9. Comparison of the quantities of ^^P transported from sheath to core in mycorrhizal roots kept {A) in aerated buffer, (B) in buffer bubbled with Nj, (C) first in buffer bubbled with Ng and then in aerated buffer Treatment Charging tinne 2 hr. 45 min. Expt total ^-P per 100 mg. dry roots Total ^^P in core Diff. after washing A. 17 hr. 45 min., air Bj. 17 hr. 45 min., Nj Bj. 41 hr. 45 min., Nj C. 17 hr. 45 min., Nj then air. Total 41 hr. 45 min. Difference for significance t S 4'4 3-O The results given in Table 9 show this to be so. A comparison of samples A with B^ and B2 of this table confirm the previous result that transport from sheath to core is greatly reduced in anaerobic conditions. Comparison of samples B2 and C shows that roots which have been kept 17! hr. in an anaerobic washing solution actively transported ^^P into the cores during a further 24 hr. in aerated solution. The loss of ^-P in anaerobic conditions did not therefore result in a total loss of that fraction of ^^P which may be transported to the core in aerated washing solutions. DISCUSSION The experiments described above show that excised beech mycorrhizas which have recently accumulated ^^P from solution, undergo after the conclusion of the period of absorption a redistribution of ^^P in their tissues. The dependence of this movement of phosphate upon temperature and oxygen supply demonstrates that it is a metabolic change and that it is not a physical process of equilibration of ^-P by exchange or diffusion. This movement of ^^p from sheath to core may be termed 'active transport'. The sensitivity to oxygen concentration of the process of active transport is comparable to the dependence of the fungus rather than that of the host upon oxygen so that it seems likely that oxidative metabolic processes in the fungus as well as accumulatory processes in the host may play a part in it. It cannot yet be decided whether the process active transport of phosphorus is as important during absorption from solutions of low phosphate concentrations as it is during the experimental conditions here used.

12 uptake of phosphate by excised mycorrhizal roots 251 During the time periods (up to 90 hr.) of the experiments in the present paper, only small quantities of ^^P are transported from sheath to core. They represent about 10% of the 3-P accumulated in the fungus, so that the absorbed phosphate in the host may only be doubled during the washing period, in the type of experiments described. The form of the progress curve of movement of '^P in aerated buffer suggests, either that the process is one which consumes a mobile fraction of phosphorus in the sheath by transporting it to the core, or that it is one which reduces a mobile fraction to a low level and is subsequently continued at reduced rate by slow formation of this fraction by phosphorus turnover in the sheath. No attempt has yet been made to distinguish between these possibilities, although the fact is of some importance in the interpretation of the function of mycorrhizal roots and in determining the quantities of phosphorus involved. The reduction of the rate of active transport in low oxygen concentrations is sharply contrasted with results of experiments upon the effect of oxygen upon the uptake of phosphorus by intact mycorrhizas (Harley et al. 1953). Although low oxygen concentrations reduce the phosphorus uptake of mycorrhizas in a charging period the amount reaching the host is relatively unaffected by them. It seems possible, tjierefore, that there may be at least two routes through the fungal sheath to the core. One through the accumulating mechanism of the fungal tissue and dependent upon adequate oxygen supply and. another by a means not so dependent upon the oxidative processes of that tissue. It is not yet certain that the former mechanism of aerobic active transport of phosphorus from sheath to core is of great importance when roots are absorbing phosphorus rapidly from solutions containing 5 mg. or more phosphorus per litre. SUMMARY 1. An account is given of experiments which help to determine a route by which phosphate passes through the fungal tissue into the host tissues of beech mycorrhizas. 2. After a period of absorption of phosphate from a solution of o-i6i mm potassium phosphate (5 mg. P per litre) in o-oi M phthalate buffer at ph 5-5, mycorrhizal roots were washed in phosphate-free buffer. '^P moved from the sheath into the core at room temperature during washing. 3. The rate of movement of ^^P from fungus to host was rapid at first but became slower after 1015 '^'' 4. The rate of movement of ^^P was temperature sensitive and became very slow at 1 C. The gio of the process of transport was high. 5. The process of transport was sensitive to oxygen concentration. It became very slow in rate when the oxygen concentration in the washing solution was below 3 %. 6. In low oxygen concentrations and in anaerobic conditions especially, ^T was lost from the roots into the washing solution at normal temperatures. No such losses occurred at low temperatures in either aerobic or anaerobic conditions nor at normal temperatures in air. The loss of ^ap from roots in anaerobic conditions was entirely from the fungal tissue and resulted from a process which had a high temperature coefficient. 7. In roots which had been returned to normal temperatures after a period of washing at low temperatures or returned to aerobic conditions after a period of washing in anaerobic conditions, transport of phosphorus from fungus to host, temporarily prevented, was resumed at an undiminished rate. 8. It is concluded that active transport of phosphorus from fungus to host occurs in

13 252 J. L. HARLEY AND J. K. BRIERLEY myeorrhizal beech roots. The mechanism of transport is dependent upon aerobic metabolic processes in the fungal tissue as well as upon the absorptive processes of the core. The phosphate which is lost from roots kept in low oxygen concentrations is entirely released from the sheath and results from temperature sensitive anaerobic processes occurring in that tissue. We wish to thank Dr C. C. MeCready with whom certain preliminary experiments upon this problem were performed, also Dr R. Scott Russell and Dr R. Martin for facilities in their Isotope Laboratory. J. K. B. wishes to thank the Agricultural Research Council for financial assistance. REFERENCES HARLEY, J. L. & MCCREADY, C. C. (1952a). The uptake of pbosphate by excised mycorrhizal roots of the beech. IL Distribution of phosphorus between host and fungus. New Phytol. 51, 56. HARLEY, I. L. & MCCREADY, C. C. (19526). 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 HARLEY, J. L., MCCREADY, C. C. & BRIERLEY, J. K. (1953). The uptake of phosphate by excised mycorrhizal roots of the beech. IV. The effect of oxygen concentration upon host and fungus. New Phytol. 52, 124. HARLEY, J. L., BRIERLEY, J. K. & MCCREADY, C. C. (1954). The uptake of phosphate by excised mycorrhizal roots of the beech. V. The examination of possible sources of misinterpretation of the quantities of phosphorus passing into the host. New Phytol. 52, 92. HARLEY, J. L., MCCIREADY, C. C. & GEDDES, A. J. (1954). The salt respiration of excised beech myeorrhizas. I. The development ofthe respiratory response to salts. (In the Press.)

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