THE UPTAKE OF PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OF THE BEECH

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1 THE UPTAKE OF PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OF THE BEECH IX. THE NATURE OE THE PHOSPHATE COMPOUNDS PASSING INTO THE HOST BY J. L. HARLEY AND B. C. LOUGHMAN Department of Agriculture, Oxford [Received 25 Janitary 1963) (With 6 figures in the text) SUMMARY Phosphate absorbed from KH2^^PO4 solutions of low eoncentration by beech mycorrhizas is rapidly esterified. After periods of a few minutes about onehalf of that absorbed is found in organic compounds. Both fungal and host tissues when exposed, after separation, to radioactive phosphate solutions show this ability for rapid esterification. Whole mycorrhizas which had been allowed to absorb KH2^^PO4 were rapidly dissected in the cold and the labelled phosphate fractions in their host and fungal tissues were separated. In the host tissue 90 % of the ^^P was found in inorganic phosphate after half a minute and this percentage fell to a little under 80 % after 10 minutes. The curve of percentage labelling of inorganic phosphate in the host tissue appeared to extrapolate to 100% at zero time. It is concluded that inorganic phosphate passes into the host from the fungus. This result is discussed in relation to previous work on phosphate absorption by mycorrhizas. INTRODUCTION The experimental work described in this paper is an attempt to decide what compound or compounds of phosphoius pass from the fungal tissue to the host in beech mycorrhizas M'hen inorganic phosphate is absorbed from an external solution. It has been previously shown that, although a direct diffusion pathway exists through the fungal sheath, it operates only in the supply of phosphorus to the host when high external phosphate concentrations are applied (Harley, McCready and Brierley, 1958). When low external concentrations (3 XIO~*M or less) of phosphate are applied, the diffusion pathway is negligibly important. The experimental results at these concentrations, which are still high compared with those expected in soil solutions, are best explained if it is assumed that phosphate is absorbed metabolically into the fungal sheath and passed from thence into the host within the living system by an active process. Clearly if it were organic phosphate which passed into the host, this would be an unequivocal piece of evidence to show that diffusive movement Avas not important. However, in the experiments described below, it was found that inorganic phosphate passed into the host, but no evidence was obtained to gainsay the previous conclusions that movement through the sheath is by processes other than diffusion. 350

2 Phosphate uptake by niycorrhiza 351 METHODS Mycorrhizal roots were collected, cleaned and sampled as described in previous work. They were exposed to labelled phosphate solutions in small conical flasks in a thermostat at 1921 C. After the absorption period, they were decanted on to a sintered filter and washed thoroughly in icecold water for several minutes. Before extraction or dissection they were rapidly blotted and the proximal 3 mm region was cut off to minimize the effect of absorption through the cut ends. Dissection was carried out rapidly upon icecold slides with cold instruments, and the material awaiting dissection for a few minutes was kept in icecold conditions. In many experiments the need for rapid manipulation necessitated the use of small numbers (six to ten) of roots in each sample. As soon as possible after treatment, roots or their separated tissues were plunged into cold HCIO4 solution (0.2 N) in small glass homogenizers. Homogenization, centrifugation, reextraction and washing of residues were all carried out in the homogenizer tube, and every effort made to keep the material cold. For extraction 1.0 ml. of 0.2 N HCIO4, and for washings 1.0 ml of 0.04 N HCIO4 were employed for samples of ten roots or less. In the one experiment where large numbers of roots were used, the material was extracted in a mortar and centrifuged in chilled tubes. The HCIO4 extract and washings were bulked, brought to ph 3.5 with ammonia and stored, if necessary, frozen solid. With large samples aliquot parts were used for various analyses. With small samples the whole extract was reduced to small volume and spotted on to chromatograms. Chromatograms were run on washed Whatman 3 mm paper for 20 hours in fer^butanolpicricwater solvent (80 ml2.2 g20 ml). After drying, the chromatograms were resolved in two ways by making Xray negatives using 2436 hour exposures and by scanning on the automatic scanner of Loughman and Martin (1957). The positions of the spots were then marked on the paper and the areas cut out and digested in 60% HCIO4 for a second estimation of radioactivity by liquid counting. These latter values are given in this paper as numerical values. The estimation of total phosphorus both in solution and in the insoluble residue was carried out by wet combustion of small quantities of dry plant material in 2.2 ml of 60% HCIO4 and 0.5 ml of cone. HNO3 followed by colorimetric estimation. The digestion of labelled areas cut from chromatograms was similarly carried out and followed by liquid counting of radioactivity. Colorimetric estimates of orthophosphate and of total phosphate after wet combustion were carried out by the method of Allen (1940) using an EEL colorimeter. RESULTS Phosphate content of mycorrhizas Table i gives results of estimation of phosphate contents of mycorrhizas. The estimates of total phosphate were obtained after wet combustion of dried samples which had been weighed. The estimates of the various fractions were obtained by extractmg similar samples of fresh material, and the sum of the fractions agreed within 6% with the direct estimates of total phosphate. The estimates for the sheath and host tissue were obtained by dissecting samples, extracting the separate tissues and estimatmg the fractions in each. j u r About 40% of the total phosphate present in mycorrhizas is found in the tungal sheath. Hence it is distributed in proportion to dry weight, for it has been shown that

3 352 J. L. HARLEY AND B. C. LOUGHMAN the sheath comprises some 39% of the dry weight of the whole (Harley and McCready, 1952; Brierley, 1953). Soluble phosphate compounds account for 3637% of the total phosphate. More than half of this soluble phosphate in whole mycorrhizas and in the host is inorganic phosphate and rather less than half in the fungal sheath. Table i. Estimates of phosphate contents of beech mycorrhizas and their constituent tissues Estimates are given for five different sampling times. At each sampling the figures are the means of five estimates. Quantities in ng P per ioo mg of dry mycorrhizas. S = fungal sheath, C = host tissue, T = total. The dry weight of sheath is 39 mg and that of the host 61 mg per 100 mg of mycorrhiza. Total P Total insoluble P Total insoluble P Soluble inorganic P Soluble organic P S 204 I C 347 T S C T S C T S C T S C T Incorporation of ^^P When mycorrhizas are immersed in dilute (io~5 M) KH2^^PO4, phosphate is absorbed and very soon the labelled phosphate appears both in the inorganic and in the organic compounds of the tissues. As shown in Fig. i, organic phosphate extracted by dilute HCIO4 in the cold, is rapidly formed and progressively becomes the major Fig. I. Uptake of phosphate, expressed as counts per minute, by beech mycorrhizas kept in 105 M KH2PO4 at 20 C. 9, total uptake; +, total soluble phosphate; x, soluble organic phosphate; D, soluble inorganic phosphate. destination of the absorbed phosphate. Later a substantial portion is incorporated in compounds which are not extracted by cold dilute HCIO4, e.g. nucleic acids and polyphosphates.

4 Phosphate uptake by mycorrhiza 353 The proportions found in these three categories (inorganic, soluble organic and insoluble) after different times of absorption are given in Table 2. After 2 minutes about 60% of the 32p is in inorganic phosphate but it decreases to about 40% after i hour. This percentage labelling is significantly lower than the percentage of total phosphate that can be extracted from whole mycorrhizas as inorganic phosphate. This is to be expected since the major part of the absorbed phosphate in these conditions is retained in the sheath, as has been repeatedly shown in previous papers. Table 2. Incorporation of ^'P in the phosphate fractions of mycorrhizal roots during a period of absorption of 1 hour in ios M KHy^POi at 19" C; thirty roots per sample Tinie (minutes) Percentage of total 'P Insoluble HCIO4 soluble Percentage of HCIO4 soluble 'P Organic Inorg; 41.SO 52 S6 61 S9 SO A primary separation of the labelled organic components of the soluble fraction may be obtained chromatographically. After 2 minutes, conspicuous peaks are present on the chromatogram scans corresponding to soluble nucleotides, sugar phosphates and perhaps phosphotriose as well as inorganic phosphate. Examples of such scans are shown in Figs. 2 and 4. In a series of experiments using samples of different numbers of mycorrhizal roots (six to thirty per sample), a reasonable consistency in the distribution of radioactivity in the various phosphate fractions after 2 minutes was observed. It become clear, however, that esterification was rapid and that successful analyses of the fractions in the constituent tissues obtained by dissection depended on rapid cooling, washing and dissection. Table 3. Incorporation of ^P into mycorrhizal roots during 2 minutes m 2 X 10 ^ M S at 21" C Samples each of a single mycorrhizal root of about lo mg fresh weight. Results given as total absorpti^on in counts/min and as percentage radioactivity in various soluble fractions. In the samples labelled 'A' total uptake directly was estimated. In the samples labelled 'B' total uptake was estimated by addition of extracted and insoluble fractions. Total 321' absorbed A B Mean Per cent ^^P extracted from samples B Per cent '^P in soluble fractions of samples B Nucleotide P Sugar P Inorganic P Hence, sample size had to be reduced to a minimum so that samples could be processed with maximum rapidity. In Table 3, analyses of six separate roots sampled at one time and treated individually are given. It will be seen that the variation is not excessive for single roots. In subsequent experiments samples of six to ten roots were employed so that rapid processing of them was possible II IS

5 354 J. L. HARLEY AND B. C. LOUGHMAN Incorporation into host and fungal tissues A series of three samples (six mycorrhiza roots each) were treated as follows. One was dissected into its fungal and host tissues, the other two were kept whole. The separate tissues and the two whole samples were allowed to absorb phosphate from labelled 2 X io^ M phosphate solution for 2 minutes at 21 C. After treatment they were washed in icecold water. One sample of whole mycorrhizas and the separated tissues of sheath and host were plunged into cold dilute HCIO4 and extracted at once. The other sample of whole mycorrhizas was rapidly dissected in the cold before extraction. Each extract, after processing, was reduced to a small volume and spotted on to paper for chromatographic separation. Fig. 2. Scans of chromatograms of soluble phosphate compounds extracted from (a) whole mycorrhiza, (b) sheath tissue, (c) core tissue after absorption from 2 " ' io"^ M ViMi^^VOi for 2 minutes at 21 C. N, nucleotide; S, sugar phosphate; P, inorganic phosphate peaks. The chromatograms were scanned and the traces of the first three samples are shown in Fig. 2. The trace of the whole mycorrhizas shows welldeveloped peaks corresponding to soluble nucleotides, sugar phosphates and inorganic phosphate. Each of the separated tissues which were exposed to phosphate separately show somewhat similar peaks. Both

6 Phosphate uptake by mycorrhiza fungal and host tissues therefore show an ability to absorb radioactive phosphate and to esterify it with similar facility. Under equivalent conditions they do not show very discrepant ratios of the quantity of radioactivity in different fractions. It is however of interest that the percentage of the radioactivity in inorganic phosphate in the fungus is 45.5 whereas that in the host is A striking contrast was seen in the comparison of fungal and host tissues separated after absorption of phosphate. As expected from previous work, the amount of radioactivity found in the fungal sheath greatly exceeded that in the host. The comparative values here obtained were not exact but about 85% was in the fungus which showed also a labelling pattern similar to that of whole roots. The host tissue, on the other hand, showed only slight evidence of the esterification of absorbed phosphate during the 2 minutes of exposure to sap. This leads to the conclusion that it may be inorganic phosphate which passes to the host from the fungal sheath during absorption. Progress of movement of phosphate to the host The combined results of two experiments showing the progress of incorporation of phosphate into the tissues of mycorrhizal roots are summarized in Fig. 3. After 30 seconds the main labelled constituent of both fungal and host tissue is inorganic phosphate. This, contains some 70% of the 32p present in the fungal sheath and about 90% of? r r (b) Minutes I Minutes Fig. 3. The percentage labelling in x, total soluble, D, soluble inorganic and, insoluble phosphate in (a) sheath and (b) core tissue after various time periods in io"^ M KH2^^PO4 at 19 C. that in the host. In both cases the proportion of radioactivity in inorganic phosphate diminishes with time, reaching about 50% in the fungus but never falling much below 80% in the host. These results are consistent with those of the previous experiments. Inorganic phosphate appears to be absorbed by the sheath and esterified in its cells to form soluble and later insoluble organic compounds. Inorganic phosphate appears to pass into the host since the curve of its intensity of labelling in that tissue may reasonably be extrapolated to 100% at zero time. Esterification takes place in the host cells but is much less evident than in those of the fungus. This conclusion is especially emphasized by an examination of Fig. 4. The scans of the chromatograms after 30 seconds' exposure to labelled phosphate are given. If that for the host extract is made at ten times the sensitivity evidence of nucleotide and sugar peaks may be obtained but it is clear that inorganic phosphate is the main labelled compound.

7 356 J. L. HARLEY AND B. C. LOUGHMAN The ability of the host to convert inorganic phosphate to organic compounds is further demonstrated by a study of the change in the proportions of labelled phosphate fractions when labelled mycorrhizas are stored in phosphatefree conditions. Whole mycorrhizas were allowed to absorb phosphate from io~5 M KH2'^^PO4 for 2 minutes. They were then kept in aerated phosphatefree solution for 30 hours. It will be seen in Fig. 5, that the inorganic phosphate in both fungus and host became esterified and ^ap appeared in soluble organic and insoluble compounds. Fig. 4. Scans of chromatograms at the same sensitivity of soluble phosphate extracts of the (a) sheath and (b) core tissues of mycorrhizas which had been allowed to absorb KH2^'PO4 for 30 seconds before dissection from lo^ M KHo^'Pa at 19" C. The peak X is scan"ned at onethird sensitivity. tb) Hours change in the distribution of labelling of phosphate compounds in fa) core and T^^a'i?? " '>;'=""hizas kept in water after a 2 minute period of uptake from io5 M u ", '^^ 19'C. n, inorganic insoluble phosphate; ;, soluble organic phosphate. It has been shown in previous papers (Harley and Brierley, 1954, 1955) that the loss]of phosphate in these conditions is small and that phosphate passes from fungus to the host, by a process dependent upon metabolic activity. It follows, therefore, that this process, called active transport to the host, is associated with the synthesis of organic compounds in the host tissue.

8 Phosphate uptake by mycorrhiza 357 DISCUSSION The results of the experiments described in this paper contribute to the knowledge of the movement of phosphate through the fungal sheath of beech mycorrhizas. The absorption of phosphate by mycorrhizas has been shown to be associated with a rapid rate of esterification so that soluble organic and insoluble compounds as well as inorganic phosphate become rapidly labelled when KH2^'^PO4 is presented. The constituent tissues, both fungal and host, exhibit the same property of esterification when separated and allowed to absorb phosphate alone. In the separated state, the host tissue is only a little less rapid in esterification than the fungus. By contrast, when whole mycorrhizas are allowed to absorb labelled phosphate the constituent tissues show different patterns. The sheath tissue not only retains the major part of absorbed phosphate, as expected from previous work, but also shows the same pattern of esterification of phosphate as in the separated condition. The host, on the other hand, still retains 80 0 of the phosphate that it receives in inorganic form even after several minutes. Indeed, the curve of percentage labelling of inorganic phosphate in the host appears to extrapolate to IOO^Q at zero time. These results lead to the conclusion that phosphate passes in inorganic form from the fungus to the host during uptake. These conclusions may be considered in the context of what has been previously discovered concerning the rate and the mechanism of movement of phosphate through the fungal sheath. In the experiments described in previous papers of this series, concentrations down to 1.6 > 105 M KH2PO4 have been employed to examine absorption and movement over periods of time from 15 minutes' to several hours' duration. Provided that the external solution does not exceed about 3x104 ^^ ^nd is aerated, uptake by mycorrhizas is linear against time for many hours and the movement of phosphate to the host, as measured by increase of radioactivity, is also linear. Mathematical treatment (Harley, Brierley and McCready, 1954) showed that this result could only be expected if the phosphate which passed from the external solution to the host tissue mixed or equilibrated with a small phosphate pool on the way. If it equilibrated with a phosphate pool whose size was of a significant magnitude compared to the total amount absorbed, then the amount of 32p appearing in the host tissue would not increase linearly with time, at least in the early stages. Instead, the increase would at first be related to some power of time, and only later would it tend to become linear. Harley, Brierley and McCready tested this theoretical conclusion in experiments in which mycorrhizas were supplied with 1.6 X 105 M KH232PO4 and the radioactivity of the host tissue measured at 15 minute intervals. Over the whole period of observations the curve of radioactivity in the host was approximately linear against time and the curve extrapolated to a point close to zero at zero time. By this means it was estimated that the phosphate passing from the external solution to the host met a quantity of phosphate in the sheath of about 0.02 ug P per 100 ^g of dry mycorrhizas. Even if this estimate is erroneous by a factor of ten or even a hundred, which is unlikely, one cannot doubt that the quantity is small compared with the lag of total phosphorus or even the 3040Mg of inorganic phosphorus found in the sheath of 100 mg of dry mycorrhizas (see Table i). Hence phosphate must pass through some region of the fungal sheath which contains a small part only of the total phosphate and of the inorganic phosphate. This region could be the protoplasmic layer within the hyphae. Loughman (i960) estimated the amounts of nucleotide and inorganic phosphate in the turnover system of the protoplasm

9 358 J. L. HARLEY AND B. C. LOUGHMAN of potato and found that they were small. Fungal hyphae may well be similarly constituted. The other possible route of passage might be by diffusion through the hyphal walls. Harley et al. (1958) obtained very strong but not absolutely unequivocal evidence that movement through the sheath from phosphate solutions was not by diffusion, except in the high concentrations of io"^ M and above. Had it been found in the present experiments that phosphate passed into the host in organic form, this would have clinched the argument. In the event, however, this was not so. Hence one must still rely on the strong but not final evidence already obtained, together with the fact that there is a metabolic movement of phosphate from accumulation sites in the sheath into the host (Harley and Brierley, 1954, 1955). When mycorrhizas were allowed to absorb radioactive phosphate for a time and were subsequently immersed in phosphatefree solutions or very dilute unlabelled phosphate, the phosphate which had accumulated in the fungus passed to the host. The process was not only dependent on oxygen supply and temperature, but was diminished in rate when phosphate was being absorbed from the external solution. External soln. Fungus Host Inorganic P Inorganic P Accumulation sites Organic P 1 t Accumulation sites Eig. 6. Suggested scheme of incorporation of phosphate into and movement of phosphate through tbe tissues of mycorrhizas. A simple scheme which would satisfy the available evidence would be that shown in Fig. 6 but further evidence is necessary to confirm it. In particular an examination of the constitution of the soluble organic phosphate compounds, especially the nucleotides, which became labelled, and a separate estimation of the protoplasmic inorganic phosphate is needed. In addition, improved technique should allow a fully quantitative study of the rate of metabolically dependent redistribution of phosphate between sheath and core after a period of absorption. ACKNO WLED GMENTS We wish to thank Mrs. J. L. Williams for able technical assistance, and the Royal Society for a grant to one of us (J.L.H.). REEERENCES ALLEN, R. J. L. (1940) The estimation of phosphorus. Biochem. J., 34, 858. BRIERLEY, J. K. (1953). Absorption of Salts by Mycorrhizal Roots of Famis svlvatica Dissertation for D.Phil., Oxford University. HARLEY, J. L. & BRIERLEY, J. K. (1954). The uptake of phosphate by excised mycorrhizal roots of the beech. VI. Active transport of phosphorus from the fungal sheath into the host tissue. New PhvtoL, 53, 240. HARLEY, J. L. & BRIERLEY, J. K. fi9s5). The uptake of phosphate by excised mycorrhizal roots of the beech. VH Active transport^of ^^P from fungus to host during uptake of phosphate from solution. Ne7v PhytoL, 54, 297. HARLEY, J. L., BRIERLEY, J_ K. & MCCREADY, C. C. (1954). The uptake of phosphate by excised mycorrhizal roots of tbe beech. V. The exammation of possible sources of misinterpretation of the quantities of phosphorus passing mto the host. Nezv PhytoL, 53, 92.

10 Phosphate uptake by mycorrhiza 359 HARLEY, J. L. & MCCREADY, C. C. (1952). The uptake of phosphate by excised mycorrhizal roots of tbe beech. III. The effect of the fungal sheath on the availability of phosphate to the core. New PhytoL, 51,343 H.ARLEY, J. L., MCCREADY, C. C. & BRIERLEY, J. K. (1958). The uptake of phosphate by excised mycorrhizal roots of the beech. VIII. Translocation of phosphorus in mycorrhizal roots. New PhytoL, 57, 353. LouGHMAN, B. C. (i960). Uptake and utilization of phosphate associated with respiratory changes in potato slices. Plant Physiol., 35, 418. GHM.'^N, B. C. & MARTIN, R. P. (1957). Methods and equipment for the study of the incorporation of phosphorus by intact barley plants in experiments of short duration. J. exp. Bot., 8, 272. H N.P.

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