THE EFFECT OF CATIONS ON THE ABSORPTION OF PHOSPHATE BY BEECH MYCORRHIZAL ROOTS

THE EFFECT OF CATIONS ON THE ABSORPTION OF PHOSPHATE BY BEECH MYCORRHIZAL ROOTS BY D. H. JENNINGS Botany Department, Leeds University {Received 30 April 1964) SUMMARY Pretreatment of beech mycorrhizal roots with relatively high concentrations (10-2 M to 10-1M) of ammonium, potassium and sodium salts can cause a reduced uptake of radioactivity from phosphate solutions containing 32p. These same salts also decrease the ability of the roots to retain radioactivity already absorbed the greatest losses occurring at low temperatures. Radioactive phosphorus both in orthophosphate and bound form is lost. Calcium ions have no effect on the absorption of radioactivity but can nullify the effect of the monovalent ions hoth on the absorption and loss of radioactivity. The results are discussed in relation to other work on cell permeability. It is concluded that the monovalent cations increase a non-specific efhux of phosphate compounds from the roots which is little affected by metabolism but which can be inhibited by calcium ions. It is also pointed out that these results have important implications with respect to a kinetic analysis of phosphate absorption from solutions of varying strength of a phosphate salt of a monovalent cation. There is evidence that monovalent cations will have a similar effect on phosphate uptake by non-mycorrhizal tissues. INTRODUCTION In general, it has been customary to consider the absorption of phosphate ions by plant cells without reference to the cation content of the medium, so that little is known concerning the eftect of this latter on phosphate absorption. This paper is specifically concerned with the effect of certain cations upon phosphate absorption by beech mycorrhizal roots. The data shows that these cations can be an important determinant of the rate of phosphate absorption in this tissue, and, furthermore, there are indications that they may affect the absorption of phosphate by non-mycorrhizal tissues in a similar manner. METHODS The methods used to obtain the data given in this paper have been described in a previous paper (Jennings, 1964). The only additional information concerns the investigation of the retention of radioactivity due to ^^p within the roots and the levels of radioactivity used. In determining the amount of radioactivity retained by the roots the following procedure was used. (i) Roots were pretreated for 5 minutes at 25 C with 5 x io-s M phosphate containing 1-20 lac 32p per ml. (ii) Roots were then transferred to the solution whose effect on the retention of radioactivity was under consideration for a variable period of time at 25 C. (iii) Roots were finally transferred either to 10-2 M phosphate or distilled water at 2 C; 20-30 ml of solution was used. The solution was only agitated continuously, by 348

Cation effect on phosphate absorption by beech 349 aeration, on those occasions when it proved necessary to determine the loss of radioactivity at definite intervals of time; i ml of solution being removed for such determinations. Otherwise the solutions were left to stand for two or tnore hours with only intermittent agitation. Table i. The phosphorus content of roots after 14 hours at 23 C in io~2 M phosphate and different concentrations of ammonium chloride Concentration of aninionium chloride 5 ^ IO"'' M lo"-' M 5 : 10 "3 M 10 ~- M 5 10"- M TO ~1 1\I Phosphorus content (mg per 100 mg dry wt.) 1.23 1.20 1.22 1.19 1.22 1.07 1.00 of roots C'u) 100 104 99 97 98 83 81 Before and after the second stage, the roots were washed four times with approximately 200 ml of distilled water at 2~ C and blotted lightly before transferred to the appropriate solution. Other experiments, concerned with the incorporation of 32p from solutions of different phosphate concentration, used levels of radioactivity varying between 0.002 and o.i nc Table 2. The effect of pretreatment with ammoniitm chloride upon the absorption of ^-P from lozv and relatively high concentrations of phosphate (fl) Pretreatment and absorption periods both 30 minutes, lg"" C Concentration of ammonium Radioactivity in roots ( o) chloride 5 / i o " 5 M 2 x i o " 3 M phosphate phosphate 100 100 5 ; ' 10 "3 jvj no 107.5 100.5 98 10-IM 95 82 92 75 {b) Pretreatment period 18 hours, absorption period 3 hours, 19^ C Concentration of ammonium Radioactivity in roots^( o) chloride 5 x 10 "^ M 10-2 M phosphate phosphate 100 100 SXIO"3M 107 94 120 94 10-IM 59 57 56 48 32P per ml depending on the concentration of phosphate and the duration of the experiment. In these experiments, the roots were dried overnight at 100 C and the radioactivity determined directly. In the experiments concerned with the retention of radioactivity, the roots were wet-ashed as previously described (Jennings, 1964) and i ml of the resultant solution used for determining the radioactivity.

350 D. H. JENNINGS RESULTS The effect of storage for 17^ hours in a range of ammonium chloride concentrations upon the absorption of ^^P over a 5-minute period from 8 X io~5 M phosphate by beech mycorrhizal roots is shown in Fig. i. Over the range 4 x io"^ M to io~^ M, ammonium chloride stimulates the absorption of radioactivity and its incorporation into all the phosphate fractions within the root. This stimulation has been described and discussed 30r 20 cn o 10 0" X 10" 10" ^ P^^treatment with different concentrations of ammonium chloride for fv " incorporation of radioactivity into the various phosphate fractions P 7 mmme h H V"'KV''''K '^^P?^"^ to 8 ^o-'mphosphate at 25 C. P,-, orthophosphate; P7, 7-minute hydrolysable phosphate; P,, phosphate stable to 7-minute hydrolysis. in a previous communication (Jennings, 1964). Above 10-2 M, absorption is inhibited, pretreatment with 10 1 M ammonium chloride giving an inhibition of 50% and a severe reduction in the amount of radioactivity incorporated into all phosphate fractions within the root. Table i shows that similar results are obtained when consideration is given to the increase of total phosphorus within the root brought about by lo'^ M

Cation effect on phosphate absorption by beech 351 phosphate in the presence of different concentrations of ammonium chloride. In this case, the accuracy of the method of chemical determination precluded the inclusion of a pretreatment period and the use of low concentrations of phosphate. Figs. 2 and 3 show that the duration of the pretreatment is important in determining the extent of the inhibition of absorption of radioactive phosphorus, inhibition increasing 50 80 Minutes Fig. 2. The effect of different periods of pretreatment with io"^ M ammonium chloride on the absorption of 32p from 5 x io"^ M phosphate over 5 minutes, both treatments at 20.5 C. in a linear manner with time of pretreatment. With short periods of pretreatment, ^^p absorption from high concentrations of phosphate is less affected; with larger periods of pretreatment, absorption from high and low concentrations appears to be equally affeeted (Table 2). Pretreatment of roots in concentrations of potassium chloride greater than io"^ M for 30 minutes at 25 C results in a decreased absorption of ^^p by the roots from 5 x io~^ M 25 r 10 20 Minutes Fig. 3. The eftect of different durations in period of pretreatment with 10 1 M ammonium chloride upon the absorption of ^^p from 5 < 10 ^^ ^ phosphate. A, pretreatment period 15^ hours, 22.5 C; B, pretreatment period 30 minutes, 19.5"' C. phosphate over a 30-minute period (Table 3). Table 3 also shows that potassium sulphate (at the same concentration as the chloride with respect to potassium), sodium chloride and sodium nitrate are as effective as potassium chloride in reducing absorption of 32p. Calcium chloride and aluminium chloride are only slightly effective. Pretreatment of roots with ammonium chloride and potassium chloride not only reduces the absorption of 32P by the roots but the same concentrations of these salts can 30

352 D. H. JENNINGS also increase the loss from the roots of radioactivity which has been incorporated previously. Loss is increased at 25 C, i.e. during the period of chloride pretreatment, and also subsequently at 2 C when the roots are transferred to 10-2 M phosphate (Figs. 4 and 5). The loss of previously absorbed radioactivity brought about by pretreatment with 10-1 M ammonium chloride has been characterized in some detail. The extent of the loss is dependent on the length of time in both ammonium chloride and in 10-2 M phosphate at 2"" C (Figs. 6 and 7). For a given period in ammonium chloride, maximum loss at 2 C is achieved after 60 minutes in 10^2 ^ phosphate, and any increase in this time brings no further change in the amount lost. On the other hand, a period of 20-35 minutes in ammonium chloride produces the largest loss of radioactivity for any one subsequent Table 3. The effect of pretreatment with various salts itpon the absorption of ^-P from 5 X io""^ M phosphate (a) Pretreatment and absorption periods both 30 minutes, 25" C Salt Radioactivity in roots ( o) KCl IO -3 M 3 > < lo" 3 M IO -'- M IO -1 M IO -4 M IO -3 M 10 _2 M 10-1 M AlCh 103 89.8 82.2 70-3 94-9 98.3 89.4 106 (b) Pretreatment period 17 hours; absorption period 50 minutes, 20 C Salt Radioactivity in roots ( o) 100 10 "^M KCl 24.0 5 X io~2 M K2SO4 20.5 io-ijvinacl 16.5 io-imnano3 19.0 io^i M CaCh 97.8 period in phosphate at 2 C. A longer period in ammonium chloride results in a slight decline in the amount lost to a value which is independent of the duration of the ammonium chloride treatment. The loss of radioactivity at 25"" C increases with increased duration of ammonium chloride treatment but the loss is never as great as that for an equivalent period at 2 C. 10-2 M potassium dihydrogen phosphate was originally used as the suspension medium for examination of loss of radioactivity at 2 C, because it was felt that maximum loss might be observed in the presence of H2PO4. Later observations showed that loss appeared to be independent of ionic composition of the medium, being as great into distilled water as into io"^ M phosphate. Sodium chloride and potassium nitrate at io-i M concentration induce losses of radioactivity of a similar magnitude to those induced by ammonium or potassium chloride (Table 4). On the other hand, the loss induced by io-i M potassium chloride can be strikingly reduced by the presence of calcium chloride in the medium (Table 5). 10-2 M calcium chloride in fact reduces the loss of radioactivity to a level below that for untreated roots. The inhibition of phosphate absorption brought about by pretreatment with 10-1 M potassium chloride is also reduced by calcium chloride, and indeed, when

Cation effect on phosphate absorption by beech 16' o 15 353 10-10" 10"' (.1 NH^Cl Fig. 4. The effect of ^ ar^ lng concentrations of ammonium chloride on the retention of radioactivity withm roots both during the treatment period of 5 minutes at 25' C (columns) and subsequently in 10 ^'- M phosphate at 2 C (circles). A, experiment of 10 October 1960; B and columns, experiment of 29 Jvlarch 1961. 30 20 c 10 M KCl 10" 10 Fig. s- The effect of varying concentrations of potassium chloride on the loss of radioactu'ity both during the treatment period of 5 minutes at 25'' C (A) and subsequently in 10 - M phosphate at 2 C (B).

354 E). H. JENNINGS this latter salt is at 10^2 ^^ absorption of phosphate is greater than when the roots are untreated. The source of the radioactive phosphorus which is lost from the roots at 2 C after 35 30 >s ^ 10- -.2 0-0 0 20 60 Minutes 80 100 120 Fig. 6. The eflfect of varying intervals of time in io "^ M ammonium chloride at 2$^ C upon the loss of radioactivity. A, loss into ammonium chloride; B, loss into io ~- M phosphate at 2 C. pretreatment with io"i M ammonium chloride has been determined from measurements of radioactivity in the various phosphate fractions within the roots before and after the regime in io~2 M phosphate at 2 C. The results (Table 6) show that radioactivity is lost 40 30.2 c o g E 5 20 10 (ii) -1 L 10 20 30 AO 45 Minutes in NH^Ct 20 AO 60 80 Minutes in K 100 120 lao 160 F'g;7- Data from one expermient showmg the loss of radioactivity into 10-2 M phosphate at 2' C. (1) 1 he effect of varying time mten'als m 10 ^i M ammonium chloride at 25 C followed by A, 10 minutes in phosphate; B, 20 minutes; C, 40 minutes; D, 60 minutes (li) The effect of varying time intervals m phosphate subsequent to (a), 15 minutes in ammonium chloride; (b) 25 mmutes; (c) 45 minutes; (d) 36 mmutes. Curve X in (i) indicates loss of radioactivity into ammonium chloride.

Cation effect on phosphate absorption by beech 355 from the orthophosphate and acid soluble bound phosphate fractions and none from the acid insoluble phosphate fraction. After 30 minutes pretreatment with ammonium chloride, something like 7.5% of the radioactivity from the orthophosphate is lost and Table 4. The effect of treatment for 5 minutes at 25'" C with various salts at io^^ M concentration upon the retention of radioactivity within replicate samples of roots both during the treatment with the salt and during a sitbsequent period in io"' M phosphate at z' C Loss of radioactivity as a j, total radioactivity Salt initially present before each treatment In salt In io~- M phosphate NH4CI 3.3 15.6 3.9 13-2 KCl 3.6 19.2 2.5 19.2 KNO3 2.8 13.4.3-3 17-5 NaCl 3.3 13-4 16.5 o of the radioactivity from the acid soluble bound phosphate is lost into 10-2 M phosphate at 2" C. After 60 minutes in ammonium chloride, the equivalent figures are 11% and i9 o- DISCUSSION The results show that pretreatment of roots with concentrations of ammonium chloride greater than 10-2 M, or with potassium chloride greater than io-^ M, causes an inhibition of phosphate absorption. Two possibilities exist for explaining the nature of this Table 5. The effect of the presence of calcium chloride during the treatment period on the loss of radioactivity brought about by treatment of replicate samples of roots for 30 minutes with io~i M potassium chloride at 25' C Loss of radioactivity as a ",, total radioactivity Solution for initially present before each treatment treatment In treatment In distilled water at 25 " C at 2 C 1.7 i-o 1.8 1-4 KCl 2.8 21.8 2.7 is-9 KCl 0.9 1-4 KCl + 10-3 MCaCk 0.8 1-4 KCl 0.7 O-74 KCl +10-2 MCaCU -87 inhibition. There might be a reduction in the rate of incorporation of phosphate into bound form, the importance of this process in phosphate absorption havmg already been indicated (Jennings, 1964), or a reduction in the rate of the primary absorption process. There are two pieces of evidence in favour of the latter possibility. First, increasing

D. H. JENNINGS inhibition of phosphate absorption with increasing concentration of ammonium and potassium chlorides is paralleled by an increasing inability of the roots to retain the radioactivity which has been incorporated. This inability to retain radioactivity is most strikingly shown at z" C, for very much more is lost at this temperature than during an equivalent period at 25' C. This very great increase in loss of radioactivity, brought about by low temperatures, indicates that the efflux of radioactivity is largely a physical process and little affected by temperature and that influx is metabolically determined and considerably affected by temperature. If this supposition is correct, the reduction in the rate of absorption (i.e. net flux) of radioactivity from phosphate solutions containing 3'-P at 25 C which is brought about by ammonium and potassium chloride will be due primarily to an increase in the rate of efflux of radioactivity. Table 6. The changes in radioactivity in the various phosphate fractions within replicate samples of roots at 2' C subsequent to treatment with io~i M ammonium chloride at 25'' C Treatment 30 minutes in NH4CI 30 minutes in NH4CI + phosphate at 2^ C Radioactivity (lo- counts/niin per Acid soluble phosphate other Orthophosphate Pi 512 528 314 299 than orthophosphate 126 128 71.0 86.6 IOO mg dry wt.) Acid insoluble phosphate 44.6 63.2 41.9 44-5 60 minutes in NH4CI 60 minutes in NH4CI + phosphate at 2 C 495 546 373 324 102 no 846 696 397 457 478 434 Second, it seems certain that monovalent cations bring about the inhibition of phosphate absorption. Calcium and aluminium ions have little effect. Indeed calcium ions clearly have an effect on the phosphate permeability of the root, which is opposite to that of monovalent cations, for calcium ions can nullify the effect of potassium chloride both on the absorption of 32p and the loss of radioactivity. Jones and Jennings (1965) have shown that sodium ions can bring about similar increases of permeability in a wide range of fungi growing in shake culture and that these increases can be prevented by calcium ions. Jennings (1963) has discussed the role of calcium in plant cell permeability. Briefly, the available evidence is in favour of the presence of pores in the outer membranes of the cells which are calcium-filled and that such calcium-fllled pores are opened in the presence of suitable concentrations of monovalent ions making the membrane more permeable. The mcrease in permeability is apparently non-specific for smaller ions or molecules. The present data are m agreement in that they demonstrate an increased permeability in the presence of monovalent ions which can be reduced by calcium ions. Furthermore, an analysis of the radioactivity which is lost at 2^ C after pretreatment of the roots with ammonium chloride shows that other acid soluble phosphates as well as orthophosphate pass into the external medium, indicating that the permeability of the cell to a number ot compounds is affected.

Cation effect on phosphate absorption by beech 357 The data are principally qualitative in nature; nevertheless, the evidence which they provide is in favour of the primary cause of the reduction of phosphate absorption brought about by monovalent ions being an increase in the efflux of orthophosphate and other phosphate compounds. However, as the pretreatment with monovalent ions is prolonged, metabolism (as evidenced by the ability of the roots to incorporate radioactivity into the various phosphate fractions) is increasingly affected, no doubt because important metabolites, certainly phosphate compounds and possibly others, are lost from the cell. It should also be emphasized that, while the data concern the effect of pretreatment of the tissue with monovalent cations upon the subsequent absorption of phosphate, they will also apply to the situation where monovalent cations and orthophosphate anions are present together in the solution external to the root. Here also there will be an increasing efflux of phosphate compounds with increasing concentration of the salt. A kinetic interpretation of phosphate absorption must take this fact into account. Furthermore, it is clear that careful attention must be given to the source of the radioactivity which is lost from the root if a quantitative study of the net flux of orthophosphate is to be undertaken. This latter stricture may well apply to a wide range of tissues, for Tanada (1955) has observed that phosphate absorption by mung bean roots is reduced by pretreatment with 5 :< 10"- M sodium chloride but stimulated when io""* M calcium nitrate is also present. There is no reason to suppose however that the concentrations of monovalent cations causing inhibition of phosphate absorption need necessarily be the same as those which are effective for beech mycorrhizal roots. Jones and Jennings (1965) showed that the sensitivity of fungi to the toxic effects of sodium ions often differed very markedly from species to species. Thus, while monovalent cations may exert a similar effect in a wide range of tissues, the similarity is not always likely to be quantitative. Some mention must be made of the maximum in the loss of radioactivity at 2" C obtained after 20-30 minutes pretreatment with ammonium chloride. It is presumed that the rise to the maximum represents the development to the full extent of the effect of ammonium chloride. The fall from the maximum in the amount of radioactivity lost suggests that radioactivity in the root is being incorporated into a form which is less easily lost from the roots. A consideration of the redistribution of radioactivity between the various phosphate fractions which occurs under these conditions (Jennings, 1964) indicates that incorporation of ^ap into the acid insoluble phosphate fraction and into the acid soluble phosphate fraction stable to 7-minute hydrolysis with 2 N HCl at 100" (P.s) and the transference of radioactivity from the smaller pool of orthophosphate to the larger pools in the sheath and the core could all be possible ways in which the radioactivity is more readily retained inside the root. All of these three processes show a decline in rate to a negligible value between 20-60 minutes after the roots have been placed in a phosphate-free medium, which is consistent with the development of a constant loss of radioactivity from the roots for all periods in ammonium chloride greater than 40 minutes. REFERENCFS JENNINGS, D. H. (1963). The Absorption of Solutes by Plant Cells, p. 204. Oliver & Boyd, Edinburgh. JENNINGS, D. H. (1964). Changes in the size of orthophosphate pools in beech mycorrhizal roots with reference to absorption of the ion from the external medium. New PhytoL, 63, 181. JONES E B G & JENNINGS, D. H. (1965). The effect of cations on the growth of fungi. (In press.) TANADA, T. (1955). Effects of ultra-violet radiation and calcium and their interaction on salt absorption by excised mung bean roots. Plant PhysioL, 30, 221.