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Differential Mobility of Lead and Zinc in Phloem Tissue of Sycamore (Acer pseudoplatanus) L.
Department of Biology, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K.
Differential Mobility of Lead and Zinc in Phloem Tissue of Sycamore (Acer pseudo platanus L.) G.
J. DOLLARD1) and N. W. LEPP
With 1 figure Received October 18, 1979 . Accepted November 16, 1979
Summary By use of a reverse flap-feeding technique, it was demonstrated that differential mobility of 210Pb and 65Z n occurred in phloem tissue of Acer pseudoplatanus 1. In all cases, 65Z n activity could be detected in the application leaf, other leaves, bark and wood of the experimental plants. 210Pb activity could only be detected in the experimental leaf, never in other tissues of the experimental plants. Further experiments demonstrated differential patterns of tangential movement of the two isotopes in Acer bark. 65Z n moved freely in this system, whereas 210Pb showed no potential for this type of movement. Key words: Trace metals, phloem transport, woody plants.
Introduction The circulation of certain trace metals in higher plants has attracted much attention; this relates to accumulation of potentially toxic elements in edible portions of fruits and vegetables, redistribution within the plant in relation to particular pollution sources, and patterns of internal circulation in the plant in relation to the overall circulation of trace metals in contaminated ecosystems. Within the plant, circulation of such metals may occur via several well-defined pathways, each possessing its own characteristics of structure, chemistry and mechanism. Additionally, certain critical transfer processes operate within or between the circulation pathways, and the barriers these transfers present to metal circulation within plants are, at best, hazily understood (HALL et ai., 1975). One such pathway which has received little critical investigation is the phloem transport system. An understanding of factors affecting trace metal movement in phloem tissue is important on two counts. Firstly, numerous metallic elements may be solubilized in rainfall (CAWSE, 1974), and an understanding of the factors affecting potential entry of such metals into sieve elements, and their subsequent transport 1) Present address Dept. of Physiology and Environmental Studies, School of Agriculture, Sutton Bonnington, Loughborough, Leics., U. K.
z. Pjlanzenphysiol. Bd. 97. S. 409-415.1980.
410
G. ].
DOLLARD
and N. W. LEPP
within these tissues, is an essential prerequisite to any interpretation of the subsequent redistribution and metabolic impact of foliar-absorbed trace metals. Secondly, reexport of metals deposited in leaves from xylem tissue can only occur via the phloem. Some knowledge of factors affecting loading and subsequent export of metals in this manner would enable the within-plant circulation patterns of certain metals to be more accurately assessed. In addition to the above points, in perennial woody species, a third aspect of trace metal movement in phloem tissue should be considered. In arborescent woody plants, trace metal deposition onto stem surfaces can occur, more frequently, in deciduous forms, at times of leaflessness. Movement of 21°Pb has been demonstrated across tree bark (LEPP and DOLLARD, 1974), and it is well established that many organic compounds, notably sugars and growth regulators, can be transported around the circumference of a stem via the bark tissue; this is so-called tangential movement (PEEL, 1964; LEACH and WAREING, 1967; LEPP and PEEL, 1971). Redistribution of trace metals in this system has received no attention. The experiments reported below arose as part of a project investigating seasonal patterns of trace metal circulation in Sycamore (Acer pseudoplatanus), growing at sites subjected to different inputs of atmospheric trace metals (DOLLARD, 1979). During the course of the work, it became clear that considerable deposition of both Pb and Zn was occurring on foliage and stem surfaces of the trees; the following experiments were an extension of this work, designed to investigate the potential mobility of trace metals in a woody plant. To facilitate this purpose, the radioisotopes 210Pb and 65Zn were employed in conjunction with previously developed techniques for assessing longitudinal and tangential movement of substances in phloem tissue.
Materials and Methods 1. Longitudinal movement This was investigated using a reverse flap-feeding technique, slightly modified from that used by CATALDO et al. (1972). As no potted material of a suitable age and size was available, samples of field-grown Acer were collected and treated in the following manner prior to experimentation. Young trees, aged 6-7 years old, were collected locally. Stems were severed approximately 5 cm above the soil surface, the cut ends immediately sealed with plastic film, and the «trees» were returned rapidly to the laboratory. Here, a 10 cm length of stem at the basal end of each «tree» was removed under water, and the remainder of the stem was then clamped in a vertical position with the cut basal end in a water reservoir. In this way, it was hoped that the formation of air embolisms in the xylem tissue would be avoided, and that the water relations of the experimental leaves would suffer no undue disturbance. This procedure resulted in the production of «trees» 1.5-2 m in height, each possessing numerous mature leaves. Four such «trees» were prepared. On each «tree», replicate leaves were treated in the following manner. A secondary vein (Fig. 1) was severed, and any attached lamina material removed to a distance of 3 mm from the cut. At the apical cut end of the vein, a microcapillary, containing 20 fll of the Z. Pjlanzenphysiol. Bd. 97. S. 409-415. 1980.
Differential mobility of lead and zinc
411
Fig. 1: Method of treatment and within-leaf sampling points for reverse flapfeeding experiments. S - microcapillary/radioisotope reservoir. 1,2, 3, 4 - areas of tissue sampled for radioisotope analysis. appropriate radioactive solution was then attached (Fig. 1). Each solution contained either 0.2,uCi/mg-1 21°Pb or 0.4 ,uCi/mg-1 65Z n, the metal being at a concentration of 100 ppm. Following this manipulation, the severed vein, and attached microcapillary, was then re-orientated to its original position, taking care in the process not to contaminate the other portions of cut vein or surrounding leaf tissue. Leaves were then left for 12 hours before sampling. Experiments with microcapillaries sealed at one end and filled with water indicated that negligible evaporation of solution occurred over this time period. In addition, in further experiments, performed as described above, rates of isotope uptake from the capillaries were measured. Distribution of both lead and zinc within the leaf were assessed according to the scheme shown in Fig. 1. In addition, the young, expanding «sink» leaves at the apex of each «tree» were assayed for radioactivity, together with bark and wood samples from this region.
2. Tangential Movement The technique used here was that used by LEpp and PEEL (1971). The only difference in procedure was the increased experimental duration of 72 hours from isotope application to bark sampling. 3. Isotope Assay Leaf, bark and wood tissues were digested for 21°Pb activity using an HCI0 4 /HN0 3 / H 2S0 4 digestion mixture, consisting of 7 : 1 : 4 70 % HCI0 4 : 97.4 Ofo H 2S04 : 65 Ofo HN0 3 • Analar grade acids were used throughout. The actual digestion process was as follows: a weighed, dried tissue sample was placed into a 20 ml plastic scintillation vial. 10 ml of the digestion mixture were added, the vials sealed and left to digest for 16 hours. Each sample was then placed in a 100 ml conical flask; at this stage, the solution was yellow. Flasks were placed on an electric hot plate and heated until the solution boiled and white fumes ap-
Z. Pjlanzenphysiol. Bd. 97. S. 409-415. 1980.
412
G.]. DOLLARD and N. W. LEPP
peared. At this point, 10 ml of de-ionised water were added. This produced a clear solution, which, on cooling, was poured hack into the original vial. The purpose of this .cfe-colouration of the solutions was to improve the efficiency of mph counting. Due to the production of a highenergy heta particle, 210Ph is particularly suited to Cerenkov counting, using water as a scintillant (JELLEY, 1958). Colour quenching and chemi-Iuminescence can reduce efficiency (HABERER, 1966; GIBSON and LALLY, 1971), so de-colouration of samples was adopted as routine. This improved counting efficiency for 210Ph as follows: Bark Wood Leaf
1.71-+ 3.80 1.84 -+ 3.44 1.67 -+ 3.49
mph aCtiVity was assayed using a Packard Tri-Carh liquid scintillation spectrometer using internal standardization. All results were corrected to D.P.M., and where required, converted to {lg of lead. 65Z n activity was assayed in an E.C.K.O. scintillation counter, using an E.C.K.O. welltype scintillation crystal of thallium-activated sodum iodide. Plant samples were counted with no prior digestion treatment, and counting efficiency ranged from 14-17010. All results were corrected to D.P.M. Results
1. Longitudinal movement The results from these experiments are given in Table 1. All within-leaf data are a mean of 31 replicated leaves for lead and 30 replicated leaves for zinc. As the D.P.M. for each element varied considerably from leaf to leaf, the within-leaf distribution of radioactivity has been expressed as a % of leaf total for each of the four sampling areas. In addition, lead and zinc content of the five youngest leaves on each «tree» is also given, together with values for wood and bark from this region. The results clearly demonstrate the apparently greater mobility of zinc in comparison with lead in the experimental system. Table 2 illustrates the relative rates of uptake of the two labelled solutions into experimental leaves. There is no difference in the rate between lead and zinc, so the differential distribution of the two isotopes subsequent to uptake cannot be attributed to different uptake rates from the microcapillaries.
2. Tangential movement Results from the tangential movement experiments are given in Table 3. Two main points emerge from these results. Firstly, lead is apparently immobile in this transport system, as in no case could any activity be detected above or below the site of isotope application. Secondly, zinc is mobile tangentially, and its pattern of movement appears to be under gravitational influence. A significantly greater quantity of zinc is found below the site of application, a situation which parallels similar findings for plant hormones in this system (LEPP 'and PEEL, 1971). Z. P/lanzenphysiol. Bd. 97. S. 409-415. 1980.
Differential mobility of lead and zinc
413
Table 1: Distribution of 210Pb and 65Z n in Acer following reverse flapfeeding application.
1. 210Pb
Ofo 210Pb distribution DPM
Fed Leaf
1 32.0
2 41.8
3 18.5
Sink Leaves Bark N.D. N.D.
4 37.7
Wood N.D.
n = 30 for fed leaf, N = 5 for sink leaves, N = 1 for bark and wood. Numbers for fed leaf correspond to sampling areas shown in Fig. 1. N.D. - not detected. 2. 65Zn
Ofo 65Z n distribution DPM
Fed Leaf
1 12.1
2 33.1
3 12.2
4 42.6
Sink Leaves 520
n = 31 for fed leaf, N = 5 for sink leaves, n correspond to sampling areas shown in Fig. 1.
Bark 337
Wood 243
= 1 for bark and wood. Numbers for fed leaf
Table 2: Mean rates of radioisotope uptake into fed leaves. Time (mins)
Isotope
164 157 Values are means of 5 replicates.
Table 3: Patterns of tangential movement of 21 0Pb and 65Z n in horizontal Acer stems. a) 21°Pb (n = 20) - No activity detected in any tissue following application. b) 65Z n (n = 20) DPM Upper 70 Source Varieties Residual Total Variance ratio
Sum of Squares D.F. 1331155.225 1 4132740.55 38 5463895.775 39
Lower 435 Mean Square 1331155.225 108756.330
Variance ratio 12.2398
= 12.2398 - significant at 5 Ofo and 1 Ofo (F) distribution. LSD = 212.953. Z. PJlanzenphysiol. Bd. 97. S. 409-415. 1980.
414
G.
J.
DOLLARD
and N. W. LEPP
Discussion
From the above results, several interesting points emerge. In the first place, distinct differences have been observed between the behaviour of lead and zinc in two distinct phloem transport pathways. There may be several reasons for the above observations. In the case of the longitudinal mobility experiments, the apparent low mobility of lead could be the result of a number of causes. Failur'e to enter the sieve element is the most obvious initial reason, but, in addition, lead which apparently does pass across the sieve element membrane may then interact with the contents of the translocation stream. Pb 2+ forms a range of insoluble compounds with the common anions found in sieve elements; chief among these being PO/- and 504 2-. This could result in preferential immobilization of any lead within the sieve-tube. In addition, lead has a high affinity for cell walls and membranes (MALONE et al., 1974), and may also bind preferentially to such structures within the sieve element. In a mass-transfer system such as undoubtedly occurs in sieve elements, no differential transport velocities will occur. Any difference in rates of movement of particular substances will reflect patterns of abstraction or immobilization along the transport conduits. This, coupled with the potential barrier at the loading stage, almost certainly accounts for the reduced mobility of lead in sieve elements. This supports the previous, tentative, conclusions of HOLL and HAMPP (1975), based on the measurement of bark lead content in field-grown Acer platanoides branches. The results obtained with zinc serve to reinforce previous observations (PATE, 1975) on the intermediate phloem mobility of this element; a sharp contrast between this essential micronutrient and the non-essential lead is inevitable. Secondly, the results from the tangential transport experiments are also of interest. The complete lack of mobility of lead in this cell to cell transport system could be a reflection of several factors. The method of isotope application, via wetted filter papers, may well be an inappropriate means of application. Lead has a high affinity for cellulose (see above) and it could well be that only a small proportion of the applied lead was actually released to the experimental material. In addition, the binding of lead to cell walls has been convincingly demonstrated (MALONE et al., 1974), and this, in combination with the factors outlined above, is almost certainly the major reason for the complete lack of lead transport in a tangential manner. In the case of zinc, a most unusual feature of its tangential transport emerges from the experiments. It has been established that in horizontal stems, the tangential transport of growth regulators is a function of gravity (LEACH and WARING, 1967; LEPP and PEEL, 1971), but no such pattern has emerged for sugars, the only other substances whose tangential transport has been investigated (LEPP and PEEL, 1971). The present results for 652n movement indicate a strong gravitationally-mediated influence over tangential redistribution. Whether this reflects a primary gravitational effect, or a secondary response mediated, perhaps, by plant hormones, remains unclear. Further investigations using other mobile cations are clearly desirable. Z. PJlanzenphysiol. Ed. 97. S. 409-415. 1980.
Differential mobility of lead and zinc
415
The main points which have emerged from these experiments are as follows: Lead and zinc show differential mobility in two phloem transport systems. There is a potential for redistribution of both elements from leaves via the phloem. Lead appears to be immobile tangentially in bark tissue, but the movement of zinc in this manner may be under direct or indirect influence of gravity. The results underline the importance of understanding the transfers between and within transport systems when considering the circulation of trace metals in whole plants. Acknowledgements G. J. D. was in receipt of an S.R.C. Research Studentship during the period this work was carried out.
References CATALDO, D. A., A. L. CHRISTY, and C. L. COULSON: Solution flow in the phloem. II Phloem transport of THO in Beta vulgaris. PI. Physiol. 49, 690-695 (1972). CAWSE, P. A.: A survey of atmospheric trace elements in the U.K. 95 pp. (1972-73). AERE-E 7669. HMSO, London, 1974. DOLLARD, G. J.: Some aspects of the behaviour of heavy metal ions in the tissue of a woody plant. Ph. D. Thesis, CNAA, 1979. GIBSON, J. A. B. and A. E. LALLY: Liquid scintillation counting as an analytical tool. A review. Analyst 96, 681-688 (1971). HABERER, K.: Measurement of beta activities in aqueous samples utilizing Cerenkov radiation. Packard technical bulletin No. 16. 14 pp., 1966. HALL, c., M. K. HUGHES, N. W. LEPP, and G. J. DOLLARD: Cycling of heavy metals in woodland ecosystems. Proc. 1st Int. Conf. on Heavy Metals in the Environment, Toronto, Canada. 2, 227-246 (1975). HaLL, W. and R. HAMPP: Lead and plants. Residue Rev. 54, 79-111 (1975). JELLEY, J. V.: Cerenkov radiation and its applications. Pergamon Press London, 1958. LEACH, R. W. A. and P. F. WAREING: Distribution of auxin in horizontal woody stems in relation to gravimorphism. Nature (Lond.) 214, 1025-1027 (1967). LEPP, N. W. and G. J. DOLLARD: Studies on lateral movement of 210Pb in woody stems. Patterns observed in dormant and non-dormant stems. Oecologia 16, 179-184 (1974). LEPP, N. W. and A. J. PEEL: Distribution of growth regulators and sugars by the tangential and radial transport systems of stem segments of willow. Planta (Berl.) 99, 275-282 (1971). MALONE, c., D. E. KOEPPE, and R. J. MILLER: Localization of lead accumulation by corn plants. PI. Physiol. 53, 388-394 (1974). PATE, J. S.: Exchange of solutes between phloem and xylem and circulation in the whole plant. In: ZIMMERMAN, M. H. and J. A. MILBURN (Eds.): Transport in Plants. 1. Phloem Transport. Encyclopaedia of Plant Physiology. New Series, pp. 451-473, Springer Verlag, 1975. PEEL, A. J.: Tangential movement of HC labelled assimilates in stems of willow. J. Exp. Bot. 15, 104-113 (1964).
Dr. N. W. LEPP, Dept. of Biology, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K.
Z. Pjlanzenphysiol. Bd. 97. S. 409-415. 1980.
Differential Mobility of Lead and Zinc in Phloem Tissue of Sycamore (Acer pseudo platanus L.) G.
J. DOLLARD1) and N. W. LEPP
With 1 figure Received October 18, 1979 . Accepted November 16, 1979
Summary By use of a reverse flap-feeding technique, it was demonstrated that differential mobility of 210Pb and 65Z n occurred in phloem tissue of Acer pseudoplatanus 1. In all cases, 65Z n activity could be detected in the application leaf, other leaves, bark and wood of the experimental plants. 210Pb activity could only be detected in the experimental leaf, never in other tissues of the experimental plants. Further experiments demonstrated differential patterns of tangential movement of the two isotopes in Acer bark. 65Z n moved freely in this system, whereas 210Pb showed no potential for this type of movement. Key words: Trace metals, phloem transport, woody plants.
Introduction The circulation of certain trace metals in higher plants has attracted much attention; this relates to accumulation of potentially toxic elements in edible portions of fruits and vegetables, redistribution within the plant in relation to particular pollution sources, and patterns of internal circulation in the plant in relation to the overall circulation of trace metals in contaminated ecosystems. Within the plant, circulation of such metals may occur via several well-defined pathways, each possessing its own characteristics of structure, chemistry and mechanism. Additionally, certain critical transfer processes operate within or between the circulation pathways, and the barriers these transfers present to metal circulation within plants are, at best, hazily understood (HALL et ai., 1975). One such pathway which has received little critical investigation is the phloem transport system. An understanding of factors affecting trace metal movement in phloem tissue is important on two counts. Firstly, numerous metallic elements may be solubilized in rainfall (CAWSE, 1974), and an understanding of the factors affecting potential entry of such metals into sieve elements, and their subsequent transport 1) Present address Dept. of Physiology and Environmental Studies, School of Agriculture, Sutton Bonnington, Loughborough, Leics., U. K.
z. Pjlanzenphysiol. Bd. 97. S. 409-415.1980.
410
G. ].
DOLLARD
and N. W. LEPP
within these tissues, is an essential prerequisite to any interpretation of the subsequent redistribution and metabolic impact of foliar-absorbed trace metals. Secondly, reexport of metals deposited in leaves from xylem tissue can only occur via the phloem. Some knowledge of factors affecting loading and subsequent export of metals in this manner would enable the within-plant circulation patterns of certain metals to be more accurately assessed. In addition to the above points, in perennial woody species, a third aspect of trace metal movement in phloem tissue should be considered. In arborescent woody plants, trace metal deposition onto stem surfaces can occur, more frequently, in deciduous forms, at times of leaflessness. Movement of 21°Pb has been demonstrated across tree bark (LEPP and DOLLARD, 1974), and it is well established that many organic compounds, notably sugars and growth regulators, can be transported around the circumference of a stem via the bark tissue; this is so-called tangential movement (PEEL, 1964; LEACH and WAREING, 1967; LEPP and PEEL, 1971). Redistribution of trace metals in this system has received no attention. The experiments reported below arose as part of a project investigating seasonal patterns of trace metal circulation in Sycamore (Acer pseudoplatanus), growing at sites subjected to different inputs of atmospheric trace metals (DOLLARD, 1979). During the course of the work, it became clear that considerable deposition of both Pb and Zn was occurring on foliage and stem surfaces of the trees; the following experiments were an extension of this work, designed to investigate the potential mobility of trace metals in a woody plant. To facilitate this purpose, the radioisotopes 210Pb and 65Zn were employed in conjunction with previously developed techniques for assessing longitudinal and tangential movement of substances in phloem tissue.
Materials and Methods 1. Longitudinal movement This was investigated using a reverse flap-feeding technique, slightly modified from that used by CATALDO et al. (1972). As no potted material of a suitable age and size was available, samples of field-grown Acer were collected and treated in the following manner prior to experimentation. Young trees, aged 6-7 years old, were collected locally. Stems were severed approximately 5 cm above the soil surface, the cut ends immediately sealed with plastic film, and the «trees» were returned rapidly to the laboratory. Here, a 10 cm length of stem at the basal end of each «tree» was removed under water, and the remainder of the stem was then clamped in a vertical position with the cut basal end in a water reservoir. In this way, it was hoped that the formation of air embolisms in the xylem tissue would be avoided, and that the water relations of the experimental leaves would suffer no undue disturbance. This procedure resulted in the production of «trees» 1.5-2 m in height, each possessing numerous mature leaves. Four such «trees» were prepared. On each «tree», replicate leaves were treated in the following manner. A secondary vein (Fig. 1) was severed, and any attached lamina material removed to a distance of 3 mm from the cut. At the apical cut end of the vein, a microcapillary, containing 20 fll of the Z. Pjlanzenphysiol. Bd. 97. S. 409-415. 1980.
Differential mobility of lead and zinc
411
Fig. 1: Method of treatment and within-leaf sampling points for reverse flapfeeding experiments. S - microcapillary/radioisotope reservoir. 1,2, 3, 4 - areas of tissue sampled for radioisotope analysis. appropriate radioactive solution was then attached (Fig. 1). Each solution contained either 0.2,uCi/mg-1 21°Pb or 0.4 ,uCi/mg-1 65Z n, the metal being at a concentration of 100 ppm. Following this manipulation, the severed vein, and attached microcapillary, was then re-orientated to its original position, taking care in the process not to contaminate the other portions of cut vein or surrounding leaf tissue. Leaves were then left for 12 hours before sampling. Experiments with microcapillaries sealed at one end and filled with water indicated that negligible evaporation of solution occurred over this time period. In addition, in further experiments, performed as described above, rates of isotope uptake from the capillaries were measured. Distribution of both lead and zinc within the leaf were assessed according to the scheme shown in Fig. 1. In addition, the young, expanding «sink» leaves at the apex of each «tree» were assayed for radioactivity, together with bark and wood samples from this region.
2. Tangential Movement The technique used here was that used by LEpp and PEEL (1971). The only difference in procedure was the increased experimental duration of 72 hours from isotope application to bark sampling. 3. Isotope Assay Leaf, bark and wood tissues were digested for 21°Pb activity using an HCI0 4 /HN0 3 / H 2S0 4 digestion mixture, consisting of 7 : 1 : 4 70 % HCI0 4 : 97.4 Ofo H 2S04 : 65 Ofo HN0 3 • Analar grade acids were used throughout. The actual digestion process was as follows: a weighed, dried tissue sample was placed into a 20 ml plastic scintillation vial. 10 ml of the digestion mixture were added, the vials sealed and left to digest for 16 hours. Each sample was then placed in a 100 ml conical flask; at this stage, the solution was yellow. Flasks were placed on an electric hot plate and heated until the solution boiled and white fumes ap-
Z. Pjlanzenphysiol. Bd. 97. S. 409-415. 1980.
412
G.]. DOLLARD and N. W. LEPP
peared. At this point, 10 ml of de-ionised water were added. This produced a clear solution, which, on cooling, was poured hack into the original vial. The purpose of this .cfe-colouration of the solutions was to improve the efficiency of mph counting. Due to the production of a highenergy heta particle, 210Ph is particularly suited to Cerenkov counting, using water as a scintillant (JELLEY, 1958). Colour quenching and chemi-Iuminescence can reduce efficiency (HABERER, 1966; GIBSON and LALLY, 1971), so de-colouration of samples was adopted as routine. This improved counting efficiency for 210Ph as follows: Bark Wood Leaf
1.71-+ 3.80 1.84 -+ 3.44 1.67 -+ 3.49
mph aCtiVity was assayed using a Packard Tri-Carh liquid scintillation spectrometer using internal standardization. All results were corrected to D.P.M., and where required, converted to {lg of lead. 65Z n activity was assayed in an E.C.K.O. scintillation counter, using an E.C.K.O. welltype scintillation crystal of thallium-activated sodum iodide. Plant samples were counted with no prior digestion treatment, and counting efficiency ranged from 14-17010. All results were corrected to D.P.M. Results
1. Longitudinal movement The results from these experiments are given in Table 1. All within-leaf data are a mean of 31 replicated leaves for lead and 30 replicated leaves for zinc. As the D.P.M. for each element varied considerably from leaf to leaf, the within-leaf distribution of radioactivity has been expressed as a % of leaf total for each of the four sampling areas. In addition, lead and zinc content of the five youngest leaves on each «tree» is also given, together with values for wood and bark from this region. The results clearly demonstrate the apparently greater mobility of zinc in comparison with lead in the experimental system. Table 2 illustrates the relative rates of uptake of the two labelled solutions into experimental leaves. There is no difference in the rate between lead and zinc, so the differential distribution of the two isotopes subsequent to uptake cannot be attributed to different uptake rates from the microcapillaries.
2. Tangential movement Results from the tangential movement experiments are given in Table 3. Two main points emerge from these results. Firstly, lead is apparently immobile in this transport system, as in no case could any activity be detected above or below the site of isotope application. Secondly, zinc is mobile tangentially, and its pattern of movement appears to be under gravitational influence. A significantly greater quantity of zinc is found below the site of application, a situation which parallels similar findings for plant hormones in this system (LEPP 'and PEEL, 1971). Z. P/lanzenphysiol. Bd. 97. S. 409-415. 1980.
Differential mobility of lead and zinc
413
Table 1: Distribution of 210Pb and 65Z n in Acer following reverse flapfeeding application.
1. 210Pb
Ofo 210Pb distribution DPM
Fed Leaf
1 32.0
2 41.8
3 18.5
Sink Leaves Bark N.D. N.D.
4 37.7
Wood N.D.
n = 30 for fed leaf, N = 5 for sink leaves, N = 1 for bark and wood. Numbers for fed leaf correspond to sampling areas shown in Fig. 1. N.D. - not detected. 2. 65Zn
Ofo 65Z n distribution DPM
Fed Leaf
1 12.1
2 33.1
3 12.2
4 42.6
Sink Leaves 520
n = 31 for fed leaf, N = 5 for sink leaves, n correspond to sampling areas shown in Fig. 1.
Bark 337
Wood 243
= 1 for bark and wood. Numbers for fed leaf
Table 2: Mean rates of radioisotope uptake into fed leaves. Time (mins)
Isotope
164 157 Values are means of 5 replicates.
Table 3: Patterns of tangential movement of 21 0Pb and 65Z n in horizontal Acer stems. a) 21°Pb (n = 20) - No activity detected in any tissue following application. b) 65Z n (n = 20) DPM Upper 70 Source Varieties Residual Total Variance ratio
Sum of Squares D.F. 1331155.225 1 4132740.55 38 5463895.775 39
Lower 435 Mean Square 1331155.225 108756.330
Variance ratio 12.2398
= 12.2398 - significant at 5 Ofo and 1 Ofo (F) distribution. LSD = 212.953. Z. PJlanzenphysiol. Bd. 97. S. 409-415. 1980.
414
G.
J.
DOLLARD
and N. W. LEPP
Discussion
From the above results, several interesting points emerge. In the first place, distinct differences have been observed between the behaviour of lead and zinc in two distinct phloem transport pathways. There may be several reasons for the above observations. In the case of the longitudinal mobility experiments, the apparent low mobility of lead could be the result of a number of causes. Failur'e to enter the sieve element is the most obvious initial reason, but, in addition, lead which apparently does pass across the sieve element membrane may then interact with the contents of the translocation stream. Pb 2+ forms a range of insoluble compounds with the common anions found in sieve elements; chief among these being PO/- and 504 2-. This could result in preferential immobilization of any lead within the sieve-tube. In addition, lead has a high affinity for cell walls and membranes (MALONE et al., 1974), and may also bind preferentially to such structures within the sieve element. In a mass-transfer system such as undoubtedly occurs in sieve elements, no differential transport velocities will occur. Any difference in rates of movement of particular substances will reflect patterns of abstraction or immobilization along the transport conduits. This, coupled with the potential barrier at the loading stage, almost certainly accounts for the reduced mobility of lead in sieve elements. This supports the previous, tentative, conclusions of HOLL and HAMPP (1975), based on the measurement of bark lead content in field-grown Acer platanoides branches. The results obtained with zinc serve to reinforce previous observations (PATE, 1975) on the intermediate phloem mobility of this element; a sharp contrast between this essential micronutrient and the non-essential lead is inevitable. Secondly, the results from the tangential transport experiments are also of interest. The complete lack of mobility of lead in this cell to cell transport system could be a reflection of several factors. The method of isotope application, via wetted filter papers, may well be an inappropriate means of application. Lead has a high affinity for cellulose (see above) and it could well be that only a small proportion of the applied lead was actually released to the experimental material. In addition, the binding of lead to cell walls has been convincingly demonstrated (MALONE et al., 1974), and this, in combination with the factors outlined above, is almost certainly the major reason for the complete lack of lead transport in a tangential manner. In the case of zinc, a most unusual feature of its tangential transport emerges from the experiments. It has been established that in horizontal stems, the tangential transport of growth regulators is a function of gravity (LEACH and WARING, 1967; LEPP and PEEL, 1971), but no such pattern has emerged for sugars, the only other substances whose tangential transport has been investigated (LEPP and PEEL, 1971). The present results for 652n movement indicate a strong gravitationally-mediated influence over tangential redistribution. Whether this reflects a primary gravitational effect, or a secondary response mediated, perhaps, by plant hormones, remains unclear. Further investigations using other mobile cations are clearly desirable. Z. PJlanzenphysiol. Ed. 97. S. 409-415. 1980.
Differential mobility of lead and zinc
415
The main points which have emerged from these experiments are as follows: Lead and zinc show differential mobility in two phloem transport systems. There is a potential for redistribution of both elements from leaves via the phloem. Lead appears to be immobile tangentially in bark tissue, but the movement of zinc in this manner may be under direct or indirect influence of gravity. The results underline the importance of understanding the transfers between and within transport systems when considering the circulation of trace metals in whole plants. Acknowledgements G. J. D. was in receipt of an S.R.C. Research Studentship during the period this work was carried out.
References CATALDO, D. A., A. L. CHRISTY, and C. L. COULSON: Solution flow in the phloem. II Phloem transport of THO in Beta vulgaris. PI. Physiol. 49, 690-695 (1972). CAWSE, P. A.: A survey of atmospheric trace elements in the U.K. 95 pp. (1972-73). AERE-E 7669. HMSO, London, 1974. DOLLARD, G. J.: Some aspects of the behaviour of heavy metal ions in the tissue of a woody plant. Ph. D. Thesis, CNAA, 1979. GIBSON, J. A. B. and A. E. LALLY: Liquid scintillation counting as an analytical tool. A review. Analyst 96, 681-688 (1971). HABERER, K.: Measurement of beta activities in aqueous samples utilizing Cerenkov radiation. Packard technical bulletin No. 16. 14 pp., 1966. HALL, c., M. K. HUGHES, N. W. LEPP, and G. J. DOLLARD: Cycling of heavy metals in woodland ecosystems. Proc. 1st Int. Conf. on Heavy Metals in the Environment, Toronto, Canada. 2, 227-246 (1975). HaLL, W. and R. HAMPP: Lead and plants. Residue Rev. 54, 79-111 (1975). JELLEY, J. V.: Cerenkov radiation and its applications. Pergamon Press London, 1958. LEACH, R. W. A. and P. F. WAREING: Distribution of auxin in horizontal woody stems in relation to gravimorphism. Nature (Lond.) 214, 1025-1027 (1967). LEPP, N. W. and G. J. DOLLARD: Studies on lateral movement of 210Pb in woody stems. Patterns observed in dormant and non-dormant stems. Oecologia 16, 179-184 (1974). LEPP, N. W. and A. J. PEEL: Distribution of growth regulators and sugars by the tangential and radial transport systems of stem segments of willow. Planta (Berl.) 99, 275-282 (1971). MALONE, c., D. E. KOEPPE, and R. J. MILLER: Localization of lead accumulation by corn plants. PI. Physiol. 53, 388-394 (1974). PATE, J. S.: Exchange of solutes between phloem and xylem and circulation in the whole plant. In: ZIMMERMAN, M. H. and J. A. MILBURN (Eds.): Transport in Plants. 1. Phloem Transport. Encyclopaedia of Plant Physiology. New Series, pp. 451-473, Springer Verlag, 1975. PEEL, A. J.: Tangential movement of HC labelled assimilates in stems of willow. J. Exp. Bot. 15, 104-113 (1964).
Dr. N. W. LEPP, Dept. of Biology, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K.
Z. Pjlanzenphysiol. Bd. 97. S. 409-415. 1980.