THE MECHANISM OF ABSORPTION AND UTILIZATION OF PHOSPHATE BY BARLEY PLANTS IN RELATION TO SUBSEQUENT TRANSPORT TO THE SHOOT

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1 THE MECHANISM OF ABSORPTION AND UTILIZATION OF PHOSPHATE BY BARLEY PLANTS IN RELATION TO SUBSEQUENT TRANSPORT TO THE SHOOT BY B. C. LOUGHMAN Department of Agriculture, University of Oxford {Received 15 February 1966) SUMMARY An attempt has been made to distinguish between the process of absorption of orthophosphate by the roots of whole barley plants and that of subsequent transport to the shoot. Such selection can be induced by treatment with growth regulators and inhibitors of specific biochemical stages in phosphorus metabolism. The presence of physiological concentrations of D-mannose in the root environment has little effect on the overall uptake of radioactive phosphate. The metabolic fate of the absorbed phosphate is, however, severely affected and the ion appears to be reversibly sequestered in the form of hexose monophosphates other than those found to be labelled under normal conditions. Both glucose-1-phosphate and mannose-6-phosphate contain radioactive phosphorus after treatment with mannose. Although overall uptake is relatively unaffected, mannose specifically inhibits further transport of the metabolized phosphate to the shoot and it is concluded that this transport is directly dependent on the prior incorporation of the incoming phosphate into a specific sequence of organic compounds. The results are discussed in relation to the possible mechanisms involved in the separate processes of absorption and transport. INTRODUCTION The mechanism of phosphate utilization by plants growing under conditions similar to those occurring in nature is now understood in broad outline, although disagreement exists between different groups of workers concerning tbe detailed steps involved. It is clearly stated by Hagen, Legget and Jackson (1957) that the process of uptake of phosphate by barley roots is chiefly linked with its incorporation into an energetically active form accompanying oxidative pbosphorylation associated with oxidation of components of the respiratory chain. Arguments can be presented to contest particular aspects of tbis thesis, but a reasonable working hypotbesis can be based on their suggestion. The uptake of radioactive phosphate by isolated root systems gives rise to labelling patterns somewhat different from those found witb whole plants, and young plants differ from old in this respect. Nevertheless in all cases incorporation of tbe phosphate into organic forms occurs witb extreme rapidity. Within 15 seconds of entry, one-third of the phosphate is found in the nucleotide fraction (Lougbman and Russell, 1957). Some evidence bas also been presented in support of the view tbat tbe process of esterification in other tissues is not necessarily essential to tbat of accumulation (Loughman, i960). The experimental resolution of this problem is difficult, but certain aspects can be tackled in an attempt to solve it. 388

Phosphate uptake and transport in barley 389 A second major problem is that of the interrelationships of the processes involved in transferring phosphate from the external environment of the root to

2 Phosphate uptake and transport in barley 389 A second major problem is that of the interrelationships of the processes involved in transferring phosphate from the external environment of the root to the extremities of the shoot. These processes may be arranged as follows: (a) accumulation of ions behind a barrier, possibly at the root surface; (b) transfer of the accumulated ions to the xylem; and (c) accumulation in the xylem and transport to the shoot. This deliberate simplification is supported by the finding that phosphorus passes to the shoot primarily in inorganic form under conditions where 80% of the recently acquired ion is incorporated into organic compounds in the cells of the root (Loughman and Russell, 1957). The question raised by this observation is whether all the phosphate entering the root passes through a stage of organic incorporation or whether that fraction passing to the shoot remains in inorganic form during passage through the root. If it is assumed that all the phosphate arriving in the xylem has passed through the metabolic pool, one must invoke a dephosphorylative step at a point before, or at, entry into the xylem in order to produce the inorganic form in the transport stream. The only available means of testing this hypothesis is by attempting to show selection between the processes of uptake and transport by the use of specific agents. This paper describes some of the results obtained with this form of experimentation. METHODS Barley plants var. Proctor were raised in an aerated phosphate-free culture solution and selected for uniformity of shoot and root size when 12 days old. Six pairs of such plants were transferred to polystyrene tanks containing 800 ml culture solution and were allowed to recover from handling for 2 days prior to transfer to tanks containing treatment solutions. After the required absorption period pairs of plants were divided into root and shoot either for counting or extraction in ice-cold o.i M HCIO4. Subsequent manipulations were carried out as described by Loughman and Martin (1957). RESULTS Effect of inhibitors on phosphate uptake and transport Wort and Loughman (1961) showed that the herbicide aminotriazole could select between the processes of uptake and transport. The metabolism of the soluble phosphate fraction of the root was unaltered by pretreatment with aminotriazole whereas a marked inhibition of incorporation of phosphate into the nucleic acid fraction occurred concurrently with a six-fold increase in the percentage transfer of phosphate to the shoot. Aminotriazole is claimed to form an aminoglycoside by elimination of phosphate from glucose-1-phosphate under physiological conditions (Fredrick and Gentile, i960). The fact that the herbicide has no effect on the incorporation of phosphate into the components of the acid soluble fraction of barley roots could be taken as evidence that glucose-1- phosphate is not involved in this process even though Jackson and Hagen (i960) consider it to be the first recognizable compound labelled during uptake of radioactive phosphate. However, we have never been able to demonstrate the formation of this amino glycoside under physiological conditions and judgement on this point must consequently be deferred. An opposite effect on the selection between uptake and transport is brought about by another herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Here again, a wide range of concentrations of 2,4-D in the external solution cause no changes in the metabolic

3 390 B. C. LOUGHMAN pattern of the root but as the concentration is increased the proportion transported to the shoot is considerably reduced (Table i). Since we know virtually nothing about the role played by these particular herbicides these observations are only valuable as an indication that selection between the processes of accumulation and transport is possible and hence a more specific approach is necessary, using agents whose metabolic function is known in more detail. Table i. The effect of pretreatment of i\-day barley plants with 2,^-dichlorophenoxyacetic acid on subsequent absorption and transport of phosphate Moles 2,4-D mfxclplant % transport Root Shoot Total Control ^ 7-3 I X i x10-= X10-* x10-* i-o ixio"' i-i i-hour uptake from IO-'M KH2"PO4 at ph 4.5 at 18 C after a 4-hour pretreatment with 2,4-dichlorophenoxyacetic acid. If a dephosphorylative step is involved prior to transfer of phosphate to the transport stream, an inhibitor of phosphatase action might be expected to have a greater effect on the transport system than on the process of absorption. A number of such inhibitors have been tested. The kinetics of inhibition by molybdate ions of the uptake of phosphate Table 2. The effect of inhibitors on absorption and transport of phosphate by i^-day-old barley plants mficlplant o transport Root Shoot Total Control IXIO'-'MDNP I X 10- 'M arsenate O5 4-7 I X 10" ^M iodoacetate I X io~^m fluoride hour uptake from 10" ^M KH2^^PO4. at ph 4.5 at 18 C. and of the acid phosphatase isolated from barley roots are very similar and there is consequently no selective inhibition of uptake or transport. Fluoride, however, at concentratrations much lower than those normally used with isolated enzymes does in fact show evidence of selection. The relevant entry in Table 2 indicates that when the absorption of phosphate by the root was reduced by only 25 % its transfer to the shoot was very severely inhibited. The magnitude of this effect is variable especially in respect of the age of the plants. This observation can be extended to other inhibitors, e.g. barley seedlings become less sensitive to 2:4 dinitrophenol and more sensitive to iodoacetate between 7 and 14 days. Effect ofmannose on phosphate uptake and transport In a number of early experiments sugars and other respirable substrates were added in an attempt to modify the endogenous metabolism. Although little effect was seen on the overall uptake, considerable changes in the metabolic pattern were noted. This effect

4 Phosphate uptake and transport in barley 301 was shown most clearly witb mannose by Jackson and Hagen (i960) and confirmation in tbis laboratory bas raised important implications concerning tbe necessity for incorporation into tbe metabolic pool prior to the transfer to tbe sboot. The presence of io~^ M mannose in the medium during uptake from io~^ M KH2 ^^P04 has little effect on the overall uptake into the root, but Fig. i shows that the distribution of activity in tbe acid soluble fraction is very different from tbat of the control plants. (a) M-6-P G-l-P (b) (c) ^ ATP UTP ADP UDP G-6-P F-6-P F-D-P PGA Orthophosphate Fig. g I. Chromatogram g scans of soluble phosphate p compounds p extracted from barley roots after f absorption bi from f IO"'M ' Kti^^VC for f i hour h at 18" C in i the h presence off ^ glucose or io~^m ^ mannose. Solvent system: tert-hutanol h l (80 ml)-"water (20( ml)-picric l i acid (2.2 g). (a) Mannose; (b) glucose; and (c) control. By contrast, the presence of glucose bas little effect on the pattern of distribution but does cause an overall increase in the amount of phosphate absorbed. Tbe major labelled components found after treatment with mannose are glucose-1-phosphate and mannose-6-phosphate, and it can be inferred that the sequential chain of reactions involved in phosphate transfer in tbe root is blocked at a step involving one or both of these compounds. Tbe relative amounts of these components varies according to the age of the plant. In the normal metabolic pattern it is clear that these substances contain little

5 392 B. C. LOUGHMAN radiodiactive phosphorus and the main sugar phosphate labelled is glucose-6-phosphate. Mannose also prevents incorporation of phosphorus into the nucleotide fractions. The special feature of the mannose effect is that the sugar must enter the root with the phosphate; pretreatment of the plants with mannose for i hour has little effect on the subsequent uptake and metabolism of phosphate. These facts suggests that the involvement must be in the primary stages of phosphate utilization and that mannose already Nucleotides G-6-P M-6-P G-l-P Orthophosphate Fig. 2. Chromatogram scans of soluble phosphate compounds extracted from barley after absorption from io-^m KHj'-POj. in the presence of mannose. (a) Control; (b) IO (c) io M; (d) io M; (e) io ^M. Relative position of radioactive compounds the sam Fig. I. accumulated by the roots cells can have little influence on the subsequent utilization of phosphate. However, a far more important implication of this phenomenon can be applied to the solution of the problem stated earlier concerning the relationship between metabolism and the fate of the absorbed phosphate. If the subsequent transport of inorganic phosphate to

6 Phosphate uptake and transport in barley r "-^- hjn" ' '=, '^ "''^<= f transport to the shoots of whole barley plants kept in IO-'M _FU4 in the presence of mannose. Symbols: A, control; o, IO-*M,-, IO-='M; I, IO"'M c Minutes Fig. 4. The time course of entry into the roots of whole barley plants kept in KH^^PO in the presence of mannose. Symbols: L, control; O, io"*!;!;, I N.P. I

7 394 B. C. LOUGHMAN the shoot does in fact depend on the prior involvement of the ion in organic combination, it is clear that the severe metabolic disturbance brought about by mannose should cause cessation of transport. On the other hand, if the ion can proceed to the shoot directly without metabolic utilization, no such inhibition should be observed. That the former situation holds is shown very clearly in Figs. 2 and 3. The effect of mannose concentration on root metabolism after a 2 hour uptake period is shown in Fig. 2 and at 10~^ M the typical pattern can be seen; the beginning of this trend is detectable at io~' M and quite clear at io""*^ M. These concentrations of mannose cannot be considered non-physiological since the sugar is so closely integrated with the normal enzymic mechanisms for available sugar utilization in plant tissues. Comparison with Fig. 3 shows that over a period of 4 hours almost complete inhibition of transport to the shoot occurs at concentration of 10" ^ M and above. It can be seen that even the higher concentrations of mannose have little eifect of accumulation into the root at a time when 50 r Fig, 5, The relationship between percentage transport of phosphate to the shoot and the mannose concentration of the root environment, the inhibition of transport is virtually complete (Fig. 4). This experiment provides the first concrete evidence that the inorganic orthophosphate passing to the shoot is first incorporated into the normal phosphorylative metabolism of the cell. The relationship between transport and mannose concentration is shown in Fig. 5. A further experiment in which 10"^ M mannose and io~^ M KH2^^P04 were fed simultaneously for 30 minutes before transfer of the plants to water indicated that the typical esterification pattern induced by mannose in the root at 30 minutes reverted over a period of i hour to the normal form, and concurrently with this change phosphate was exported to the shoot. Another very significant point is that under conditions of steady

Phosphate uptake and transport in barley 395 state uptake and transport of phosphate the addition of mannose to the external solution brings about an immediate cessation of transport.

8 Phosphate uptake and transport in barley 395 state uptake and transport of phosphate the addition of mannose to the external solution brings about an immediate cessation of transport. DISCUSSION The fact that selection between processes of uptake and transport can be modified by external agents is not surprising but it is of interest that the range of such agents is so diverse. Chemical agents of both specific and unknown modes of action as well as a number of environmental changes such as temperature, light intensity and oxygen tension can alter the balance between the two processes and these will be discussed in later publications. The intrinsic interest of these phenomena lies in the possible integration of the biochemical and physiological problems involved in the utilization of ions by plant tissues. The most clear-cut separation of the processes involved in phosphate uptake is shown by the experiments involving the use of mannose as the differentiating agent. It is known that this sugar can inhibit the grovrth of roots when supplied over a number of days (Stenlid, 1957), but the immediate nature of the response in the short-term experiments described here can hardly be associated with this known effect of mannose. Reference to Figs. 2 and 3 indicates that io~* M mannose has a significant effect on both metabolism and transport, yet concentrations of this order must be considered physiologically possible in many plant tissues. At the present time, our knowledge of the process of phosphate transfer through a series of intermediates of high energy potential during oxidative phosphorylation is insufficient either for assessment of the significance of the observations or to speculate on the biochemical implications of the mannose effect. The enzymes involved in the interconversion of hexoses through their phosphates can be demonstrated in extracts of barley seedlings and it is obvious that, under favourable conditions, mannose can be produced at the expense of glucose or fructose. However, the fact that pretreatment with mannose has no effect on subsequent phosphate uptake argues against any hypothesis which involves the in vivo production of mannose as a means of modifying the destination of absorbed phosphate. The presence of phosphoglucose-isomerase is readily demonstrated in extracts of the roots used in these experiments whereas the activity of phosphomannose-isomerase is very much lower. The absence of the latter enzyme from tomato roots has been used by Goldsworthy and Street (1965) to explain the inhibition of root respiration by mannose, and they further state that mannose-6-phosphate builds up in the tissue thereby immobilizing the cytoplasmic phosphate which results in a lowering of the respiratory rate. The fact that pretreatment of barley seedlings of low phosphate status with 10"^ M mannose has little effect on subsequent entry of phosphate suggests that no significant change in cytoplasmic levels of phosphate has occurred. Any mannose-6-phosphate present is therefore probably in the vacuole since its presence in the cytoplasm might be expected to inhibit enzymes whose substrate is glucose-6-phosphate. Since glucose-6- phosphate is a normal component in the sequential labelling pattern, the absence of any pretreatment effect with mannose argues against vacuolar mannose-6-phosphate being the causative agent. On the other hand, the action of a phosphatase could lead to the recycling of any phosphate converted to mannose-6-phosphate at an earlier stage, i.e. at the point of entry into the tissue. This is borne out by the observation that recovery of the phosphorus transport is relatively rapid after transfer of the plants to a solution containing no mannose.

9 296 B. C. LoUGHMAN Mannose and phosphate must be provided simultaneously for the effect to manifest itself, and there is some indication that part of the phosphate is held in the form of glucose-1-phosphate even though some conversion of mannose to mannose-6-phosphate occurs. Under these conditions, low concentrations of mannose-6-phosphate could inhibit phosphate incorporation by disturbance of the equilibrium between glucose-6- phosphate and glucose-1-phosphate. If the phosphomannose-isomerase activity is low, thus preventing the transfer of phosphate from mannose-6-phosphate through the normal glycolytic and oxidative pathways, then an overall build up of mannose-6-phosphate is likely. Consequently, although the overall uptake is unaffected, the distinct process of subsequent transport can be completely blocked. If the incorporation of phosphate into organic forms is a necessary prerequisite for the subsequent transfer of the inorganic ion to the xylem a hydrolytic step is required to release orthophosphate from organic combination. Since this orthophosphate would he a product of the organic esters previously formed, its specific activity would be, at least during the early stages of uptake, lower than that of the esters from which it arises. At first sight the data presented by Jackson and Hagan (i960) appear to confirm this view. Reference to Table i of their paper shows that after a 10 minute uptake period the specific activity of the orthophosphate in the root is approximately 0.5% that of the external medium whereas the specific activities of the two identified compounds claimed to he precursors, uridine diphosphoglucose and glucose-1-phosphate, are 2.7% and 6.9% respectively. However, the value for the specific activity of the inorganic phosphate fraction is determined by extraction of the tissue and must therefore involve the total inorganic phosphate of the tissue when the calculation is made. It has been demonstrated (Loughman, i960; Bieleski, 1963) that the orthophosphate of the plant cell can be distributed between the vacuole and the functional metabolic pool in a ratio of at least 20 : i. Allowing for the presence of 5 % of the total cellular orthophosphate in a turnover system, this must increase the specific activity of the phosphate in this turnover system to about 10% of the external medium after 10 minutes, rather than the figure of 0.54% quoted hy Jackson and Hagan, and at all subsequent times the specific activity of this orthophosphate will be higher than that of the other reactants in the metabolic pool. Resolution of the complex problems involved in considering hypotheses of the kind discussed here can only be achieved by comparison of accurately determined specific activities of the reactive compounds within the root and in the xylem transport system. Accurate assay of the latter is difficult owing to the presence of phosphate already in the stem tissues prior to the arrival of the new supply of ion, which might be released into the xylem during manipulation involved in sampling. Recent experiments have shown the possibility of tackling this problem by means of specialized techniques with individual plants and the results of such experiments will be reported in a subsequent paper. REFERENCES BIELESKI, R. L. (1963). Turnover rates of phosphate esters in fresh and aged shces of potato tuber tissue. PL PhysioL, Lancaster, 38, 586. GoLDSWORTHY, A. & STREET, H, E. (1965). The carbohydrate nutrition of tomato roots. VIII. The mechanism of the inhibition by D-mannose of the respiration of excised roots. Ann. Bot., N.s., FREDRICK, J. F. & GENTILE, A. G. (i960). The formation of the glucose derivative of 3-amino-i,2,4- triazole under physiological conditions. Physiologia PL, 13, 761. HAGEN, G, E,, LEGGET, J. E, & JACKSON, P. C. (1957), The sites of orthophosphate absorption by barley roots. Proc. natn. Acad. Sci., U.S.A., 43, 496. JACKSON, P. G. & HAGEN, G. E, (i960). Products of orthophosphate absorption of barley roots. P/. Lancaster, 35, 326.

10 Phosphate uptake and transport in barley 397 LoUGHMAN, B. C. (i960). Uptake and utilization of phosphate associated with respiratory changes in potato slices. PI. PhysioL, Lancaster, 35, 418. LouGHMAN, 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, jf. exp. Bot., 8, 272. LouGHMAN, B. C. & RUSSELL, R. S. {1957). The absorption and utilization of phosphate by young barley plants. IV. The initial stages of phosphate metabolism in roots. J'. exp. Bot., 8, 280. STENLID, G. (1957). Comparison of the toxic effects of some sugars upon growth and chloride accumulation in young wheat roots. Physiologia PL, 10, 807. WORT, D. J. & LOUGHMAN, B. C. {1961). The effect of 3-amino-i,2,4,-triazole in the uptake, retention, distribution and utilization of labelled phosphorus by young barley plants. Can.jf. Bot., 39, 339.

THE UPTAKE OF PHOSPHATE BY EXCISED MYCORRHIZAL ROOTS OF THE BEECH

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