THE UPTAKE OF PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OF THE BEECH

Transcription

1 [296] THE UPTAKE OF PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OF THE BEECH VII. ACTIVE TRANSPORT OE ^^P FROM FUNGUS TO HOST DURING UPTAKE OE PHOSPHATE EROM SOLUTION BY J. L. HARLEY AND J. K. BRIERLEY Department of Botany, Oxford University (Received 9 September 1954) (With I figure in the text) A previous paper (Harley & Brierley, 1954) has described a movement of phosphate from fungus to host in excised mycorrhizal roots of the beech which is dependent in rate upon oxygen supply and temperature. The movement was described as active transport. Active transport was observed after a period of absorption of labelled phosphate from an external supply when roots were washed in phosphate-free buffer solution. Hence the ^^P transported to the host during the washing phase was drawn from ^^P accumulated by the fungal tissues during a previous period in labelled phosphate. It was not certain whether the particular type of active transport described above took place while roots were absorbing phosphate from an external supply, and the present paper deals with this problem. METHODS Similar methods to those described by Harley & Brierley (1954) were used in the work reported here. The experiments usually consisted of two parts, the charging stage and the washing stage. In the former excised roots were placed in labelled potassium phosphate solution (5 mg. P per litre) buffered at ph 5-5 in o-oim-phthalate, and in it the roots were agitated at 25 C in a thermostat for 1-4 hr. In the latter, the washing stage, roots which had been charged were transferred to either phosphate-free buffer or buffer containing unlabelled potassium phosphate. The quantities of phosphorus entering the host during the washing phase are expressed in arbitrary units. The reasons for this will be found in Harley & Brierley (1954). RESULTS Fig. I summarizes an experiment in which roots were charged for 3 hr. in labelled phosphate in a buffer solution containing 5 mg. P per litre. Graph A shows the quantity of *T present in the cores of mycorrhizal roots after washing in phosphate-free buffer and in buffer containing different concentrations of phosphate. At each point on the curve the mean quantity of ^-P in the cores and the standard error of the dissections is given. The movement of *T to the host is progressively reduced in rate as the concentration of phosphate in the washing solution is increased. The maximum rate of

2 of phosphate by excised mycorrhizal roots 297 movement occurs in phosphate-free buffer. Graph B in Fig. i shows that during the whole period of washing no significant loss of previously accumulated 32p from the roots occurred, a result which agrees with those of previous experiments (Harley & McCready, 1952; Harley & Brierley, 1954; Harley, McCready & Geddes, 1954). Table i sets out the results of several experiments which confirm the results expressed in Fig. I, and show that the movement of ^T to the host becomes very slow when roots are washed in solutions containing above i mg. P per litre. Table 2 summarizes the results Amount of P in washing solution (mg./l.) Fig. 1. A comparison of the quantities of '^P in arbitrary units actively transported from fungus to host when charged roots are washed in phosphate-free buffer and buffer containing phosphate. Charging time 3J hr. Washing time 17J hr. Graph A: quantity of "-P in cores of 100 mg. dry roots calculated from mean of estimates of total ^^P for that treatment. Standard errors of estimate of quantity in core made by dissection are plotted for each point. Graph B: solid symbols; total quantities of '^P in 100 mg. dry roots after washing (replicates). Table i. The effect on transport of ^^P of washing charged mycorrhizas in a series of buffer solutions containing different quantities of phosphorus Charging times 4, 3J and 4 hr. respectively. Washing time i8, 17 and 21 hr. respectively. Amounts of '^P in cores calculated from the mean of estimates of total '^P for that treatment. Estimates of ^^P in cores in arbitrary units. Exp. no. Control (unwashed) 0 P cone, in washing solution (mg./l.) O'O S * * ±o-i7t 7-O ±0-34 S O-3I * Pooled dissections. -f- Standard error of mean.

3 298 J. L. HARLEY AND J. K. BRIERLEY of three experiments which show that when roots are switched from a buffer solution containing 5 mg. P per litre (B, Table 2), where the rate of transport of ^^^P is slower than that in phosphate-free buffer (A, Table 2), to a buffer solution containing no phosphate, movement of ^^p to the host is increased (C, Table 2). The reduction in the rate of ^^p transport when roots are kept in unlabelled phosphate is not due to a mobile fraction Table 2. The results of three experiments which show the effect on active transport of switching mycorrhizas charged with ^T from solutions containing unlabelled phosphate to phosphate-free buffer Experimental conditions. A: Charged roots placed in phosphate-free buffer. B: Charged roots placed in buffer solutions containing 5 mg. P per litre. C: As condition B then switched to phosphate free-buffer. D: Charged in unlabelled phosphate and switched to labelled phosphate parallel with B to estimate P during the washing period of B. Charging times 3, 3 and 3^ hr. respectively. Washing times as m table. Results expressed in arbitrary units of ^''P per 100 mg. dry roots. Exp. no Control unwashed 36-7 Core A Pooled dissections ^^P in cores of treatments B Washing time 19 J hr Washing time i8i hr Difference between core means for significance ^ = 0-05 = 1-25 p = o-oi = i-68 Washing time 18 hr. C As B then I9i hr. in C 6-1 As B then 24 hr. in C 5-2 As B then 24 hr. in C =2P in treatment D Core time 22f hr time 21J hr Pooled dissections labelled with ^^P being utilized in the fungal tissues but is due at least in part to a diminution of those reactions which transport ^^P from accumulated phosphorus in the fungal tissue to the host, while phosphate is being absorbed from an external source. In the experiments ^^'^ (Table 2) the quantities of phosphorus absorbed during the period of washing in phosphate solution were estimated by means of parallel samples kept in solutions labelled with ^^P. The total phosphorus absorbed and the quantities passing to the core are shown in Table 2 D. As expected, considerable quantities of phosphate were absorbed, and the quantity passing to the core greatly exceeded that moving to the core in any of the other treatments. We can discern therefore the following processes. When an external supply is present phosphate passes to the sheath and through the sheath tissue to the core. When no external supply is present some of the phosphate which has been accumulated in the

4 of phosphate by excised mycorrhizal roots 299 sheath passes to the core. This latter process is diminished v^^hen a low external supply of phosphate is present and is sensitive to temperature and oxygen supply as has been previously shovi^n. The rate of transport of ^^P by this process falls with time of washing and ultimately reaches a low value (Harley & Brierley, 1954). This decrease in rate might be attributed to a reduction in the quantities of phosphate compounds available for the process. Table 3 summarizes the results of two experiments which support this view, for a second period of charging in labelled phosphate causes an increase in the rate of active transport during a second washing period. It is probable that part of the phosphate accumulated in the fungal cells is in a form readily available to participate in an oxidative metabolic process which results in a net movement into the core. Table 3. Two experiments demonstrating the effect of recharging mycorrhizas after a washing period in phosphate-free buffer First charging periods z\ and 3^ hr.; first washing periods 21 hr. Second charging periods 2J and 3i hr.; second washing periods 21 and 24 hr. respectively. Results expressed in arbitrary units of ^''P per ioo mg. dry roots. Exp. no p in core p in core ist charging ist washing nd charging 2nd washing Washed throughout after ist charging I DISCUSSION The experiments described in this paper carry the problem of the movement of phosphorus from fungal tissue to host tissue a stage further than previous experiments. It was first suggested (Harley & McCready, 1952) that during absorption phosphorus might pass through the sheath either by diffusion through interhyphal spaces of the fungal tissue or by way of the living cells of the hyphae. Later Harley & Brierley (1954) showed that there was a process dependent on oxidative metabolism and sensitive to temperature by which phosphorus accumulated in the fungal tissue passed to the host if the roots were kept in phosphate-free buffer solutions. The possibility of leakage of phosphorus from the fungal tissue was studied, and it was found that in aerated solutions no significant leakage occurred at any temperature and that in low oxygen concentrations where leakage from the fungus into the external solution occurred, no significant transport to the host tissue was observable. It was concluded therefore that a movement, called active transport, from the fungal tissue into the host tissue, occurred within the living system and depended in rate upon a metabolic process itself dependent on oxygen supply and temperature. In that paper, as in the present experiment, no method was found for expressing the quantities of phosphorus moving into the host in weight units. The results were expressed in arbitrary units based upon the specific activity of the solution used during the charging period. These arbitrary units represent minimum estimates in jug. per 100 mg. of dry roots

5 3OO J. L. HARLEY AND J. K. BRIERLEY of the amounts of phosphorus concerned in active transport. If the specific activity of these phosphate compounds is lower, as it may well be, than that presented during the charging period these minimum estimates may well be very much lower than the actual quantities moving. The amount by which the real values exceed the estimates may indeed vary with the time of the experimental period and the conditions in which movement is studied. We can be sure, nevertheless, that the movement must be from a site of accumulation in the sheath cells into the core, for the total volume of the sheath is about 25 % of the whole root volume (Harley, McCready & Brierley, 1953) and could contain at most only 0-9 [Mg. of phosphorus in solution at the same concentration as the external solutions. The estimated amount transported during the washing period is many times this quantity in most of the experiments described. Moreover, if the source of phosphorus for active transport were that in the free spaces of the sheath, leakage of phosphorus into the washing solution would presumably be detectable. The experimental study of active transport in the presence of low phosphate concentrations has shovirn that the rate of movement of the labelled phosphate from the sheath to the core is reduced when phosphate is being absorbed from the external solution into both sheath and core. This reduction may represent a real diminution or cessation of the movement of that phosphate fraction which had been labelled in the sheath. If this is true the process can be represented as a single route of movement through the living cells of the fungus: P accumulated external PX sheath Pc core In the protoplasmic region of the sheath phosphate reacts with some metabolic product, produced in maximum quantity in the presence of adequate oxygen supply, to form compound PX. This intermediate takes part in the phosphate metabolism of the sheath tissue and from it or its products phosphate is also passed to the host and to the accumulating sites in the fungus. When the external supply of phosphate is removed or diminished, utilization of the accumulated phosphate in the sheath occurs, leading to a net movement of phosphate from sheath to host. In such a case the rate of movement will depend essentially upon processes leading to the availability of X (i.e. oxygen supply and temperature), or when this is in excess, on the availability of external phosphorus or accumulated phosphorus. This mechanism would provide an adequate explanation of the available experimental results, and pending a more detailed analysis of the compounds concerned seems a satisfactory working hypothesis. It visualizes the constant proportion of sheath and host absorption during from low phosphate concentrations as arising from a utilization of a common source of phosphate PX, and is consistent with the probability that direct diffusion of phosphate through the free spaces of the sheath to the host tissue is very small in low external concentrations of phosphate. Whatever the mechanism of movement of phosphate through the fungus to the host certain points of ecological importance have already been raised by this work. First, high rates of accumulation of phosphate by beech mycorrhizas have been demonstrated. As

6 of phosphate by excised mycorrhizal roots 301 an accumulating system mycorrhizal roots are more able to compete with micro-organisms of the litter zone than uninfected roots. Secondly, although the primary site of maximum accumulation is in the fungal tissue the active transport mechanism is potentially capable of maintaining a movement of phosphate from that accumulated in the fungus to the host tissue. Brierley (1955) has shown that the conditions of oxygen supply in the natural habitat of beech mycorrhizas are such as to allow rapid active transport to occur. SUMMARY 1. Experimental evidence is set out which shows that active transport of *^P from fungus to host is reduced in rate when roots are washed in buffer solution containing phosphate. When roots which have been returned to phosphate-free buffer after a period in buffer containing phosphate, active transport, temporarily reduced, is resumed at a rapid rate. 2. Phosphate charged roots kept in phosphate-free buffer until the rate of transport had reached a low level showed an increased transport rate after they had been charged a second time. 3. It is suggested that the phosphate absorbed from the external solution competes successfully for a substance produced in respiratory metabolism so that phosphate accumulated in the sheath is not mobilized. In the absence of external phosphate supply the phosphate already accumulated in the sheath is utilized. Movement of phosphorus into the core is mainly from the external solution when this contains phosphate, but is from the sheath phosphate when the external supply fails. REFERENCES BHIERLEY, J. K. (1955)- Seasonal fluctuations of the concentrations of oxygen and carbon dioxide in the litter' layer of beech woods with reference to salt by excised mycorrhizal roots of the beech. J. Ecol. (in the Press). HARLEY, J. L. & BRIERLEY, J. K. (1954). The of phosphate by excised mycorrhizal roots of the beech. VI.'Active transport of phosphorus from the fungal sheath into the host tissue. New Phytol. 53, 240. HARLEY, J. L. & MCCREADY, C. C. (1952). The of phosphate by excised mycorrhizal roots of the bee'ch. II. Distribution of phosphorus between host and fungus. Nezv Phytol. 51, 56. HARLEY, J. L., MCCREADY, C. C. & BRIERLEY, J. K. (1953)- The of phosphate by excised mycorrhizal roots of the beech. IV. The effect of oxygen concentration upon host and fungus. Neiv Phytol. 52, 124. HABLEY, J. L., MCCREADY, C. C. & GEDDES, A. J. (i9s4)- The salt respiration of excised beech mycorrhizas. I. The development of the respiratory response to salts. Nezv Phytol. 53, 427.

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