Zinc tolerance and the binding of zinc as zinc phytate in Lemna minor. X-ray microanalytical evidence

JPlantPhysiol. Vol. 137.pp. 140-146{1990}

Zinc Tolerance and the Binding of Zinc as Zinc Phytate in Lemna minor. X-Ray Microanalytical Evidence R. F. M. VAN STEVENINCK!, M. E. VAN STEVENINCK2 , A. J. WELLS!, and D. R. FERNANDO! 1

2

School of Agriculture, La Trobe University, Bundoora, Victoria, Australia Botany Department, La Trobe University, Bundoora, Victoria, Australia

Received March 15, 1990· Accepted June 7,1990

Summary Electron probe X-ray microanalysis of fracture faces of quench frozen hydrated bulk samples of Lemna minor fronds exposed to high levels of Zn (300 I'M) has shown the presence of globular deposits up to 1 I'm in diameter containing Zn, K and P in parenchyma cells of mature fronds, and Zn, K, Mg and P in immature parenchyma cells of daughter fronds (or turions). Although this method of direct analysis of bulk samples does not lend itself to a quantitative comparison of number and composition of globules in Zn-tolerant and Zn-sensitive clones of Lemna minor, it has the advantages of simplicity of specimen preparation, rapid detection of Zn-P-containing globules, and avoidance of diffusional artifacts. Comparisons with prepared K Mg phytate and Zn standards, and additional data obtained by X-ray microanalysis of freeze-substituted thin sections, established the elemental proportions of Zn, Mg, K and Ca relative to P, which indicate that generally up to 8 (molar ratio of Zn to phytic acid = 4) and sometimes up to 12 (molar ratio of Zn to phytic acid = 6) valencies can be occupied by Zn and up to 4 by K and/or Mg. Some of the Ca that appears to be associated with globules may result from elemental redistribution during the process of freeze-substitution especially when Ca oxalate crystals are present in neighbouring raphide cells. X-ray microanalysis provided evidence for the absence of Zn from these raphides.

Key words: Lemna minor, X-ray microanalysis, zinc·containing globules, zinc oxalate, zinc phytate, zinc tolerance.

Introduction Van Steveninck et al. (1987 a) showed that roots of Des· champsia caespitosa when challenged with high levels of zinc (up to 1000~) in nutrient solution have the capacity to bind a substantial amount of zinc in globular deposits. These globules occurred mainly in small vacuoles of the expanding cortical cells of the root elongation zone and appeared identical to the globoid crystals of phytate characterised by Lott et al. (1984, 1985). It was later shown by means of electron probe X-ray microanalysis of freeze-substituted tissue that these globular bodies in high zinc roots of Des· champsia caespitosa may contain a high percentage of zinc (20.1 % by weight), while further quantification and comparison with artificially prepared phytic acid standards con© 1990 by Gustav Fischer Verlag, Stuttgart

firmed that the globules consisted of metal complexes of phytate in which up to six of the twelve valencies are occupied by zinc and the remaining valencies by magnesium and potassium (Van Steveninck et al., 1987b). Phytic acid (myo-inositol hexakisphosphate) is generally known to play an important role in the storage of minerals in plant reproduction (seeds, pollen tubes, storage roots), but is rarely found in normal vegetative tissues (Cosgrove, 1980). Although electron probe X-ray microanalysis has shown that the principal minerals stored as phytate are magnesium and potassium (Lott, 1975; Lott et al., 1982), traces of heavy metals such as iron (Lott et al., 1982) or manganese (Buttrose, 1978; Lott, 1984) have been detected by this means. Hence, it seemed worthwhile to determine whether the binding of excess zinc by phytic acid as a possible detoxifica-

Zinc tolerance in Lemna minor tion mechanism may have wider significance in the plant kingdom. This aim is best achieved by testing plant species with a known capacity for phytic acid synthesis in vegetative organs. Aquatic plants, such as Lemna gibba and Wolffiella floridana, have been reported to have such a capacity (Roberts and Loewus, 1968; Scheiner et al., 1978), and small aquatic plants have a further advantage as they are easily exposed to a toxic range of heavy metals under controlled conditions. Thus it was relatively simple to establish clones of Lemna minor expressing substantial differences in zinc tolerance. The previous detection and analysis of globular deposits of zinc phytate in Deschampsia caespitosa depended on the complex and time-consuming technique of freeze-substitution of tissues with an organic solvent, followed by epoxy resin embedment and then thin sectioning which often resulted in damaged sections due to the extreme hardness of the globular deposits (Van Steveninck et al., 1987 b). Hence, in addition to freeze-substitution, the potential advantage of using a more rapid and simple method, i.e. direct analysis of the fracture face of bulk frozen hydrated samples, was explored.

Material and Methods Clones resulting from multiplication of individual fronds obtained from locally grown Lemna minor L. were tested for Zn tolerance at concentrations of 100, 300 and 1000!£M ZnS04 added to halfstrength modified Johnson solution of the following composition: 3 mM KNO), 2 mM Ca(NO)h, 1 mM NHJ-hP0 4, 0.5 mM MgS0 4, 25 ~ KCI, 12.5 ~ H)BO), 1 I'M MnS04, 1 I'M ZnS04, 0.25!£M CUS04, 0.25 ~ H 2Mo0 4 and 25 ~ NaFeEDTA. The solution was renewed twice per week. Two clones were selected for this study: clone A (Zn-tolerant) which showed only a slight growth inhibition at 3oo!£M Zn, and clone B (Zn-sensitive) which showed severe growth inhibition at 100 ~ Zn and no growth, necrosis and ultimate death of fronds at 3oo!£M Zn. Plants were grown under controlled environment conditions of 8/16 h light/dark, 25/22°C light/dark temperature and 220!£mol m -2 s- 1 Iight intensity. Plants were generally harvested for X-ray microanalysis after 1 week of exposure to treatment solutions containing 1 ~ Zn (control) or 300 ~ Zn in half-strength Johnson solution. Freeze-substitution Fronds were cut into three or four pieces with a sharp razor blade and plunged into a mixture of 8 % methyl cyclohexane in 2-methyl butane at -180°C. They were subsequently transferred to the substitution fluid (dry acetone or 2 % OS04 in dry acetone with activated molecular sieve added), sealed, and maintained at -72 °C with gentle shaking for 4 days. Samples were brought to room temperature over 2 days and gradually infiltrated with Spurr's medium. All transfers were carried out in a moisture-proof cabinet through which dry nitrogen gas was circulated. Electron probe x.ray microanalysis A. Frozen-hydrated samples in the scanning mode (SEM): One or two mature fronds of Lemna were placed (with no added liquid) in a wedge-shaped slot in a 1.0 cm diameter carbon stub, quench frozen in melting nitrogen (-210°C), and fractured with a cold (-180 0C) scalpel blade before insertion in the cold stage (-190°C) of a scanning electron microscope aSM840) by means of

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a Hexland CTl000 cryo-transfer system. In order to enhance structural detail within cells, the specimens were etched (differential removal of ice through sublimation) by temporarily (1-2 min) raising the cold stage temperature to -85°C. X-ray data were obtained using a TN 5500 X-ray analyzer at an accelerating voltage of 15kV with a monitored probe current ranging from 0.3 to 0.6 nA, producing a dead time of 25-30% from 100sec live-time collection of spectra. Spectra were usually collected in the reduced screen mode at 10,000 times magnification resulting in scan areas of approximately l!£m2 • B. Freeze-substituted samples in the transmission mode (TEM): Single sections (approximately 150 nm thick) were cut dry and mounted on G75 pioloform-coated nickel grids. Subsequently, grids were transferred by means of a specially constructed graphite receptacle onto the liquid nitrogen-cooled transmission stage of a JSM840 scanning electron microscope equipped with a transmission detector (TED 40). X-ray data were obtained at an accelerating voltage of 20kV with a probe current in the range of 0.1-0.3 nA giving detector dead times of approximately 25 % for 100 sec of live time spectrum collection. All spectra were analysed by defining appropriate regions of interest which were set at the points where the smoothed peak joined the background continuum, i.e. at 1.14 -1.34 keV or 1.12 -1.42 keV depending on the size of the Mg K.,. peak, 1.82-2.18keVor 1.78-2.26keV depending on the size of the P K.,. peak, 3.14-3.50keV for K K a , 3.50-3.88keV for Ca K.,., 0.86 -1.13 keV or 0.86 -1.16 keV depending on the size of the Zn La peak and 8,44-8.86keV for Zn K.,.. A relatively wide region of interest was selected for large peaks in order to optimize the number of net counts. Ca Ka net counts were determined after deconvolution of the overlapping Ca K.,. and K Kil peaks. C. Standards: Phytate standards were prepared from the di-magnesium tetra-potassium salt of phytic acid (M~~CJi60W>6, Sigma) by applying a small quantity of the fine crystalline material to double-sided tape attached to a 1.0 cm diameter carbon stub. Randomly chosen, small sized, crystals (1- 2 I'm) were analysed in the reduced screen scanning mode (SEM) at x 10,000 magnification under the same analytical conditions (probe current, voltage, take-off angle, magnification, scan area) as those used for the analysis of cellular globules in the SEM mode. Additional standards were prepared by mixing ZnS04 solution in colloidal graphite to give final concentrations ranging from 250 to 1000 mM ZnS04 in 9 % colloidal graphite. Immediately after agitation with a vortex mixer, the suspension was applied to a 0.25 mm hole in the center of a carbon stub, quench frozen in melting nitrogen and fractured under the same operating conditions of the SEM as those used for the analysis of the plant material.

Results The fracture faces of frozen Lemna leaf tissue generally show only the position of cell walls and the presence of intercellular spaces (Fig. 1 A). Little structural detail of the cell's interior can be observed. Under these circumstances, however, it is possible to locate Zn-containing deposits by means of an X-ray mapping program, especially when the more intensive X-ray emission of P is used as an indicator to show the presence of globular deposits (Fig. 2 A and B). Generally, the X-ray emission of Mg (Fig. 2 C) is either absent or too weak to allow recognition of deposits in the scanning

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R. F. M. VAN STEVENINCK, M. E. VAN STEVENINCK, A. J. WELLS, and D. R. FERNANDO

Fig. 1 A and B: Fracture faces of frozen hydrated Lemna frond tissue. Bars represent 10 /Lm. A: Maintained at - 190°C and showing very little detail of cellular contents. B: Briefly etched at - 85°C and subsequently maintained at -190°C. Differential removal of ice provides details of cell structure, but also creates an uneven surface topography. Note the presence of a large Zn-containing globule (arrow) in a parenchyma cell adjacent to a small vein (v).

mode, while the general abundance of K provides too high a background to allow the detection of K-containing deposits. Another disadvantage of operating in the scanning mode is the high magnification that is required to make detection of deposits possible by this means. Hence, only a region of a single cell can be scanned at one time, and it would take many hours to complete the scanning of a complete crosssection of a Lemna frond. More structural detail of the cells can be seen after differential sublimation (<
Fig. 2 A, Band C: Characteristic X-ray maps of a particular cell region at 10,000 x magnification. A: Representing X-rays emitted by P. B: Representing X-rays emitted by Zn. C: Representing X-rays emitted by Mg. necessary to compare these spectra with those obtained from single K Mg phytate crystals of similar size with pre-determined chemical contents of 4.8 % Mg and 13.8 % K by weight (ex Sigma) (Fig. 3 D). These percentages imply an elemental ratio for Mg/K of 0.565 and Mg1.60K2.83C6H902J>6 as the mean chemical formula of the standard. Because of the difference in counting efficiency for the different elements in the K Mg phytate standard, multiplication factors were derived in order to adjust the X-ray analytical

Zinc tolerance in Lemna minOT

Fig. 3 A to F: Electron probe X-ray microanalysis spectra obtained at 10,000 x magnification and a reduced scan area of 1JLm2. A - E in SEM mode; F in TEM mode. A: From a globule in an immature cell of a daughter frond of a Zn tolerant Lemna plant exposed to 300 JLM Zn. Frozen hydrated tissue. Note presence of Zn, Mg, K and P. B: From a globule in a mature cell of a Zn tolerant Lemna frond exposed to 300JLM Zn. Frozen hydrated tissue. Note presence of Zn, K and P and absence of Mg. C: From a globule in a mature cell of a Zn tolerant Lemna frond exposed to normal nutrient solution (1 JLM Zn). Frozen hydrated tissue. Note presence of Mg, K and P. D: From a small crystal of K Mg phytate (ex Sigma). Note presence of Mg, K and P. E: From portion of a Ca oxalate crystal in a raphide cell of a mature frond of a Zn tolerant Lemna plant exposed to 300 JLM Zn. Frozen-hydrated tissue. Note absence of Zn. F: From portion of a Ca oxalate crystal in a raphide cell of a mature frond of a Zn sensitive Lemna plant exposed to 300 JLM Zn. Freeze-substituted, epoxy embedded, thin section. Note absence of Zn.

A

B

c

D

E

F

results to match the chemical analysis data. Values for elemental ratios based on chemical analysis and by application of these multiplication factors to X-ray analytical data are shown in Table 1. It is necessary to apply a factor of 0.89 x to the net counts for K and 2.41 x to the net counts for Mg. These factors, which were obtained for bulk samples in the SEM mode at 15 k V, were similar to the values used earlier (0.97 x for K and 2.49 x for Mg) for thin samples in the TEM mode at 20kV (Van Steveninck et aI., 1987b). Therefore a correction factor of 4.24 x was applied to Zn La counts to achieve equivalence of net counts for Zn and P (Van Steveninck et aI., 1987 b). These values were used to calculate elemental ratios of Zn, Mg, K and Ca with respect to P in globular deposits, which are summarized in Table 2. A high level of Zn in the treatment solution causes a significant displacement of Mg from globular deposits in the mature frond tissue of both the Zn tolerant (clone A) and the Zn sensitive clone (clone B), while Mg remains an important component in the immature daughter frond tissue (Table 2). Globular deposits with a high P content in material that was not exposed to Zn (control) were extremely rare in clone A and were not detected at all in clone B material. The low level of K in the control treatment (Table 2) was unexpected and could not be verified because of the scarcity of globular deposits with a high P content. Contrary to this, globular deposits were relatively frequent especially in cells near conductive tissue of mature fronds exposed to high levels of Zn and in some of the parenchyma cells of immature daughter fronds. Although the frequency of deposits in clone A appeared to be much higher than in clone B, the non-random distribution of deposits and their detection by means of scanning electron microscopy of random fracture faces, pre-

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Table 1: Elemental ratios of K, Mg and P in a K Mg phytate standard sold as MgzK.C 6H 602Jl6 but with actual contents of 4.8 % Mg by weight and 13.8 % K by weight, compared with elemental ratios determined by X-ray microanalysis of small crystals (means of 10 analyses ± standard error of mean) in SEM mode at 15 kV and 0.03 nA beam current. Elemental ratios Chemical analysis X-ray analysis* X-ray analysis** (given contents) KIP 0.472 0.472 ± 0.010 0.515±0.011 Mg/P 0.267 0.266±0.010 0.275±0.010 Mg/K 0.565 0.575±0.032 0.541±0.030 * Multiplication factors required to achieve ratios obtained from chemical analysis data when analysing bulk samples of phytate in the SEM mode at 15 kV: 0.89 for K, 2.43 for Mg. ** Using multiplication factors previously determined for thin samples of phytate in the TEM mode at 20 kV: 0.97 for K, 2.49 for Mg (Van Steveninck et aI., 1987 b).

cluded any attempts to accurately determine the relative numbers of deposits. The low values and variability of the Zn KaIZn La ratios also provide a strong indication that the elemental ratios given in T able2 cannot be accurate. The generation of high energy Zn Ka (8.39-8.91 keY) X-rays may become inefficient through «charging» which results from poor electron conductivity of uncoated frozen bulk samples, especially at 15 k V when the kinetic energy of the electrons in the beam becomes marginal for the production of high energy X-rays. This is illustrated by the fact that in fractured frozen standards (SEM mode) of 250-1000 mM ZnS04 in 9 % graphite, the value for Zn KaIZn La is reduced from 4.04±0.20 at 20kV to 1.57±0.14 at 15kV (=3.90 at 20kV for thin sec-

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Table 2: Mean elemental ratios of the metals Zn, Mg, Ca and K with respect to P and ratios of net Zn Ka and Zn La counts based on X-ray microanalysis of globular deposits in etched, frozen bulk samples of mature and immature (daughter) fronds of a Zn tolerant (A) and a Zn sensitive clone (B) of Lemna exposed to 300 J.!M Zn over a period of approximately 1 week (n indicates number of analyses). Clone and Zn treatment

n

Elemental ratios Mg*/P

Zn*/P

Ca*/P

K*/P

Net count ratio Zn Ka/Zn La

0.07±0.005 0.30 ±om Clone A, Control 9 0 0 0.76±0.065 0 0.64±0.005 Clone A, 300 mM Zn, mature 10 0 0.65±0.08 Clone A, 300 mM Zn, immature 10 0.31±0.03 0.39 ±0.03 0.005 0.23±0.02 1.01±0.27 0.013±0.007 Clone B, 300 mM Zn, mature 6 0.79±0.07 0 0.51±0.05 0.45±0.13 0.17±0.03 0.37 ±0.03 Clone B, 300 mM Zn, immature 13 0 0.19±0.02 1.60±0.52 * Zn, Mg, Ca and K counts multiplied by 4.24, 2.49, 1.0 and 0.97 respectively to adjust for differences in counting efficiency with respect to P (Van Steveninck et al., 1987 b).

Table 3: Mean elemental ratios of the metals Zn, Mg, Ca, K with respect to P, metal content (atoms of Zn, Mg, Ca, K per molecule of phytic acid), and number of valencies occupied by metals per molecule of phytic acid in globular deposits of mature and immature frond tissue of a zinc tolerant Lemna clone exposed to 300 J.tM Zn as determined by electron probe X-ray microanalysis of thin sections in the TEM mode at 20 kV (n indicates number of analyses for each of 5 different fronds, a-e). Age of tissue

n

Zn*/P

Elemental ratios Ca*/P Mg*/P

K*/P

Zn

Metal content of phytic acid Mg Ca K

Valencies occupied by Zn, Mg, Ca and K

0.33 4.32 0.34 0.72 0.056 0.05 0.30 1.98 11.9 a. Mature 9 0.36 5.88 0.006 0.30 0.04 1.80 2.16 17.6 b. Mature 0.98 8 0.06 0.25 3.51 0.92 0.36 1.50 0.59 0.153 c. Immature 11.1 8 0.16 3.00 0.280 0.08 1.68 0.48 0.98 11.3 d. Immature 5 0.50 0.12 4.26 0.47 0.073 0.13 0.78 0.72 11.8 e. Mature 4 0.71 * Zn, Mg, Ca and K counts multiplied by 4.24, 2.49, 1.0 and 0.97 respectively to adjust for differences in counting efficiency with respect to P.

tions in TEM mode, VarI Steveninck et al., 1987 b). The graphite starIdards have a smooth surface and produce relatively steady values for the Zn K,,/Zn La ratios, whereas with arI etched specimen the extreme roughness of the surface topography may unfavorably affect the detection of Zn La (0.88-1.17keV) and other low energy X-ray emissions such as Mg Ka (1.16-1.36keV). Thus exact ratios of Zn, Mg, Ca arid K to P CarInot be determined in this type of material, and calculations such as metal content and number of valencies occupied by metals per molecule of phytic acid (VarI Steveninck et al., 1987 b) were not attempted. Although the probability of diffusion artifacts cannot be entirely excluded even by this method, the next step was to carry out a program of freeze-substitution (Van Steveninck et al., 1987 b) aiming to further characterise the globular deposits induced by high levels of Zn in Lemna. TabId gives elemental ratios based on net X-ray counts obtained from thin sections with TEM at 20kV. Unfortunately, the epoxy resin infiltration of frond tissue was not satisfactory in clone A material, mainly due to problems encountered because of the presence of large intercellular spaces in the fronds. However, the results obtained with clone B material confirm to a large degree that the globular deposits consist of phytate, because calculation of the metal content of deposits (atoms of Zn, Mg, Ca and K per molecule of phytic acid = P-6 myoinositol), arid further estimates of the number of valencies occupied by metals per molecule of phytate (TabId) indicate with one exception that 11- 12 valencies per molecule are occupied by Zn, Mg, Ca and K. The results also confirm the relatively higher Mg content of globular deposits in immature tissue (daughter fronds) than in mature frond tissue. It should also be noted that with freeze-substitution there was a tendency for some Ca to be present that was not

detected by means of direct X-ray arIalysis of bulk frozen tissue. In fact, in one case when the level of Ca was particularly high, the ratio of metal valencies per molecule of phytate was raised to a value as high as 17.6, which seems to indicate a diffusion artefact or the presence of material with a high Zn arid/or Ca content in the immediate vicinity of the characteristic phytate globules. On the other hand, in spite of many repeated analyses of the crystalline deposits in raphide cells, Zn was never detected in Ca oxalate crystals in frozen hydrated material (SEM mode, Fig. 3 E) nor in freezesubstituted epoxy embedded material (TEM mode, Fig. 3 F). The high ratios of Zn to P that were recorded in frozen hydrated mature tissue (SEM mode, Table2) were fully confirmed by analysis of freeze-substituted thin sections (TEM mode, TabId). Discussion X-ray analysis of fracture faces of frozen hydrated bulk tissue has enabled Zn-containing globular deposits to be detected in parenchyma cells of floating fronds of Lemna minor (duck weed) after exposure to high levels of Zn (300 pM). The same technique was also used for the detection and arIalysis of Ca oxalate crystals in raphide cells. Unfortunately, fracture faces show little or no detail of cellular structure, and in order to overcome this problem it is necessary to briefly raise the cold stage temperature to - 85°C and apply a certain degree of etching (differential removal of ice) to the specimen (Echlin et al., 1982). However, under these circumstances any qUarItitative assessment of the concentration of a particular element by comparison with prepared frozen standards of known concentration is no longer reliable

Zinc tolerance in Lemna minor (Echlin and Taylor, 1986), although claims to the contrary have been made by authors who have applied a reproducible and equal degree of etching to both the standards and specimen material (Stelzer et a1., 1988; Koyro and Stelzer, 1988). This, of course, is unlikely to be the case when the degree of hydration and density of the mineral deposits varies from that of the standards, in particular, in crystalline deposits versus solutions. Charging, which reduces the kinetic energy of the electrons producing the X-rays, and an uneven topography, which results in high variability of take-off angle for the detection of X-rays, also produce problems in quantitation that are expressed in the wide range of values for Zn Kal Zn La ratios (Table 2). These problems typically occur when using bulk samples, so quantification based on ratios of net counts is not reliable and precludes the determination of exact ratios of Zn, Mg, Ca and K to P. However, the method is useful for the rapid detection of globular mineral deposits and for qualitative analysis such as presence or absence of Mg in globules formed in immature and mature frond tissue. The more complex and time-consuming procedure of freeze-substitution and epoxy embedding was necessary for an accurate characterisation of the mineral contents of the globular deposits. Although mineral redistribution and diffusion artifacts cannot be entirely excluded by this method, these artifacts are less likely to occur with relatively insoluble biomineralized material than with soluble ions (e.g. K + , Na +, CI-) in vacuoles. But, it should be noted that the high density globules are easily lost from thin sections because they are difficult to cut (Van Steveninck et a1., 1987 a, b). With the exception of a group of data obtained from one frond, the mineral element ratios that can be derived from the X-ray data have consistently indicated a total of close to 12 valencies occupied by Zn, Mg, Ca and K per molecule of phytic acid (Table 3). One might argue that the individual globules could consist of a conglomerate of Zn, Mg, Ca and K ortho-phosphate, but the consistency of the ratios obtained for each frond and the high degree of substitution of Mg by Zn in mature frond tissue indicates that a complex molecule such as phytic acid must be involved in binding the 4 metals. Furthermore, globule formation from highly soluble components such as Mg or K ortho-phosphate appears extremely unlikely, whereas Zn phytate complexes with more than two molecules of Zn per phytate (ZnD phytate species with n = 3 to 6) have been shown to be insoluble at neutral pH (Nolan et a1., 1987). Polyphosphate granules of a similar size which occur frequently in lower forms of plant life (Chlorella, Micrococcus, Tetrahymena, Cyanophyceae, etc.) have been shown to contain significant amounts of K, Mg, Ca and in some cases Zn, and therefore it has been suggested that they function not only in P storage but also as a source of essential metals (Baxter and Jensen, 1980). These authors have also shown by means of X-ray microanalysis that these polyphosphate bodies are capable of sequestering Cd, Co, Cu, Hg, Ni, Pb and Zn with equivalent losses of Mg and K and therefore suggested that polyphosphate bodies may provide cells with a mechanism of removing metals present in a toxic range of concentrations Oensen et a1., 1982). The X-ray spectra presented in this study show a marked resemblance to those obtained for the polyphosphates. However, the presence of

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polyphosphates in higher plants is yet to be established, whereas phytic acid is recognized as a major phosphorus and metal storage constituent in higher plants (Cosgrove, 1980). Nevertheless, attention should be drawn to the striking analogy between polyphosphates in lower plants and phytate in higher plants. Finally, the unusually high level of sequestration of 17.6 metal valencies per molecule of phytate, which was recorded after freeze-substitution in one particular frond of Lemna, requires further comment. Evidently, two structures of phytic acid are possible (Cosgrove, 1966): i.e. 12 dissociable hydrogen ions per molecule can be shown by potentiometric titration in aqueous solution, while 18 can be detected in glacial acetic acid solution (Brown et al., 1961). Thus it seems possible that in cells with a particularly high Ca content, the process of freeze-substitution may lead to some mineral redistribution and therefore a higher degree of metal binding, or alternatively, but this seems to be an unlikely explanation, regions of phytate and Ca from Ca oxalate may have been superimposed during analysis. However, the possible occurrence of the latter type of artifact is excluded when microprobe analysis is carried out on bulk frozen hydrated tissue. Hence, the evidence for Zn binding by phytate is unequivocal and the two methods must be regarded as complementary for the analysis of biomineralized structures in plant cells. Similarly, evidence has been presented showing that the system that controls accumulation of excess Ca through the rapid formation of Ca oxalate in rap hide cells (Franceschi, 1989) is specific for Ca and has no role as a possible mechanism for detoxification of excess Zn. Further work comparing Zn-tolerant and Zn-sensitive clones at various levels of P in the nutrient medium is envisaged. Acknowledgements Support by a grant from the Australian Research Council is gratefully acknowledged.

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