Plant Physiol. (1979) 64, 954-9580032-0889/79/64/0954/05/$00.50/0

Transport of Divalent CationsCATION EXCHANGE CAPACITY OF INTACT XYLEM VESSELS

Received for publication April 27, 1979 and in revised form July 18, 1979

SIEBE C. VAN DE GEIJN AND CHARLES M. PETIT"1 2Association Euratom-Ital, P.O. Box 48, 6700 AA Wageningen The Netherlands

ABSTRACT

The cation exchange capacity of the intact xylem vessels in cut shootsof papyrus (Cyperus papyrus spec.) has been determined. The cationexchange capacity is independent of the cation concentration in thetranspiration stream, and is equal for Ca and Co. The high value of thecation exchange capacity (0.6 to I x 10-7 equivalents per square centimetervessel wall surface) leads to the hypothesis that the porous structure of thevessel wall, and not only the inner vessel wail surface, acts as a cationexchanger.

Differences between anion (132Plphosphate, 145CaIEDTA2-, IllICdmI-EDTA2-), and cation (145Cal2, I.ll.Cdm'I+) movement are explained interms of transport with the transpiration flux or by exchange reactions.The competition between exchange sites and natural or synthetic ligandsfor the divalent cations is discussed.

detectors (15, 19). Results of perfusion experiments with the samematerial are included.The transport characteristics ofthe divalent cations (Ca2+, Cd2+)

are compared with those of the anionic complexes (CaEDTA2,CdEDTA2-) and [32P]phosphate.

MATERIALS AND METHODS

DETECTORS AND ELECTRONIC EQUIPMENT

The electronic equipment used for the count rate measurementshas been described before (18). Up to four semiconductor detec-tor/amplifier chains can be used simultaneously to record theresponse with time of the count rate at various positions along thestem (Fig. 1) after a supply of the radioactively labeled solutions.The detectors have a circular sensitive surface, (25 mm2), and arecoilimated to admit only the fl-radiation from about 7 mm ofstem.

The role of adsorption and exchange processes in uptake as wellas translocation of cations in plants has long been recognized (2,6). The movement of Ca has been the subject of many investiga-tions (1, 2, 5, 8, 9, 15, 16). The exchangeability of Ca with otherdivalent cations (1, 5, 16) as well as the increased mobility of Cain the presence of chelating agents have been demonstrated (5, 8,9). In this respect the xylem cylinder has been suggested tofunction as an exchange column in which the carboxyl groups ofpectic substances in the cell walls form the exchange sites (11).

Values for the CEC3 of plant tissues, particularly roots (4, 6),have been reported and a possible relationship with the relativeuptake of monovalent and divalent cations has been discussed(20). In most instances, the reported values concern the CEC ofthe bulk material irrespective of their relation to the uptake andtransport processes.To our knowledge the CEC of the intact transport system in

plant stems has never been measured. Such quantitative infor-mation, however, is very important for a better understanding ofdistribution processes of cations, especially Ca, in plants andrelated physiological disorders.The present paper reports the CEC of the xylem vessels, meas-

ured in intact shoots of papyrus (Cyperus papyrus spec.) for a widerange of solution concentrations of Ca and Cd. The principle ofthe method has been described elsewhere (19).Use has been made of radioisotopes 45Ca, ll5Cdm, 32p, and the

in situ measurement of their radiation by solid state radiation

' Present address: Department of Soil Science, Catholic University ofLouvain, Place Croix du Sud 2, B-1348 Louvain-la-Neuve Belgium.2The grant of the International Agricultural Centre, Wageningen,

making possible the stay at the Associations' Institute for Application of

Atomic Energy in Agriculture is gratefully acknowledged.3Abbreviation: CEC: cation exchange capacity.

RADIOISOTOPES

Sources of llSCdm (0.1-1 mCi/mg Cd in 0.1 M HCI), 45Ca (10-40 mCi/mg Ca as CaCl2), and 3P (30-100 Ci/mg as Pi in diluteHCI) were purchased from The Radiochemical Centre, Amer-sham, U.K. The penetration power of the fl-rays of ll5Cdm (Ema= 1.63 MeV) is comparable to that of 32P (E.. = 1.71 MeV),allowing a direct comparison of the measurements without ab-sorption corrections.

PLANT MATERIAL AND SET-UP

Papyrus plants (C. papyrus spec.) were grown in a compostmixture in 15-liter pots in a greenhouse and amply supplied withwater. Shoots (about 1 m long; 0.8- to 1-cm diameter) were excisedand quickly cut again under water to prevent air entry into thetransport vessels. After the transfer to a climate-controlled growthchamber (20 C; 60-70%o RH; light 35 w/m2) the shoots wereacclimatized to the changed conditions for 1 h in deionized H20.Three semiconductor detectors were placed at equal distances (25cm) along the stem, the lower one at about 15 cm from the cutend (Fig. 1). Transpiration was measured continuously by weigh-ing the experimental solutions. Water and ion uptake proceededat the same rate and the ionic concentration in the experimentalsolution remained constant with time.

In perfusion experiments the leaf rosette was cut and a 30-cm-long stem segment retained. The solutions were supplied to thebasal end and the apical end was connected air-tight to a nozzleand by a thin tubing to the cap of an exchangeable liquidscintillation vial. Reduced pressure (8 x 104 Pa) was applied to asecond nozzle at the cap of the vial. This rather strong suction wasnecessary to compensate for the "air leakage" through the aeren-chym of the stem.The perfusate was collected during successive periods of time,

weighed, and analyzed for tracer content by liquid scintillationcounting (45Ca) or Cherencov counting ("15Cdm).

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CATION EXCHANGE CAPACITY OF XYLEM VESSELS

FIG. 1. Set-up for measurements of transport in cut shoots of papyrus.Three detectors are positioned along the stem, the fourth one at a leaf.Four preamplifiers can be seen at the left. A digital balance is used tomeasure the transpiration.

The pH of all experimental solutions was adjusted to 7, theapproximate pH of the xylem sap, with KOH or HNO3.The total number of vessels, as well as the circumference and

cross-sectional area of the vessels, were determined from measure-ments on fresh cross-sections in the light microscope. Stem di-ameter as well as the dimensions of the vessels varied with thematurity of the plants. The number of vascular bundles in a stem,however, was rather constant. For a stem with a diameter of 1 cma typical example would be: 120 to 150 bundles with each 2 vesselsof 70- to 90-pm diameter in the central part, and another 100bundles with 200 vessels of 20- to 60-pm at the periphery of thestem. In our experiments the vessel cross-sectional area variedfrom 8 X 10-3 to 2.5 x 10-2 cm2 and the circumference from 7 to12 cm.

EXPERIMENTAL PROCEDURE

Anions and Anionic Complexes, Intact Shoots. Shoots weresupplied at the basal end for various periods of time (5 s-60 min)with a 115Cdm-labeled solution (1-2.5 ,Ci/ml; 1-3 x 10-4 M Cd)containing excess Na2EDTA (10-2 M). The same shoots weresupplied with [32P]phosphate (KH2PO4) for comparison. Eachlabeling period was followed by a treatment with deionized H20.CEC Determinaton, Intact Shoots. The shoots were treated

with a ll`Cdm-labeled solution (0.5-1 ,uCi/ml) at various totalCd(N03)2 concentrations (I0-4_10-2 M). The duration of the sup-ply was sufficiently long to reach the phase in which a low andconstant rate of increase of the measured count rate at the lowerdetector position was established (see below). At that time thesolution was replaced by deionized H20, until a new steady-state

was reached. Subsequently the shoot was supplied with an unla-beled Ca(NO3)2 or Cd(NO3)2 (10-2 M) or Na2EDTA (10-2 M)solution until a new equilibrium was established. The shoot wasthen offered a 1ll5CdmJEDTA solution (excess EDTA) and finallytransferred to deionized H20. The duration of each treatmentdepended both on the Cd concentration in the applied solutionand on the transpiration rate.CEC Deternination, Stem Segments. In the perfusion experi-

ments the sequence of treatments described above for the experi-ments on intact shoots was essentially maintained. However, inaddition to the experiments with labeled Cd solutions (5 x 10-3M Cd(N03)2) a second series was done using Ca instead of Cd,and 'Ca as a tracer, at a total Ca(NO3)2 concentration of 5 x l0-3M.

RESULTS

Anions and Anionic Complexes, Intact Shoots. Short duration(5 s-2 min) application of [32P]- or [ll5CdmJEDTA-labeled solu-tions to the cut stem of a papyrus in all cases resulted in thepassage of a "wave" of tracer along the stem (Fig. 2). Thepropagation speed of this wave can be determined from the timelag of the passage of the peak at the successive detector positions.The speed of 3P was equal to that of [ll5CdmJEDTA2- and bothwere always higher than the mean speed of the bulk water,calculated from the transpiration rate (5-15 ml/h) and the meas-ured xylem vessel cross-sectional area (0.015-0.025 cm2). Thedifference between measured and calculated speed (about 30%o)was explained by the existing differences in the rate of watermovement in the individual vessels, as determined experimentallywith a dye.

Application of [`5Cdm1EDTA for a prolonged period (Fig. 3;20 min), showed that an initial rapid increase in count rate wasfollowed by a steady-state phase, in which only a slow increasecould be observed. At this steady-state phase apparently all xylemvessels were filled with the labeled solution, and the remainingslow increase could be ascribed to the much slower processes ofequilibration with the surrounding tissues, and irreversible accu-mulation. This was confirmed by the effect of a "wash" withdeionized H20; the count rate dropped rapidly to a low residuallevel which represented the short term irreversibly bound fractionof the "5Cdm. From these observations it was concluded that both[32Plphosphate and the anionic complex 1ll5Cdm]EDTA2- weretransported with the bulk transpirational water, in agreement withthe observations of Heine (7) for [3PJphosphate.CEC Deterninaton, Intact Shoots. The response with time of

the ll5Cdm count rate in the initial loading phase (non-steady-state

tun (mmn)FIG. 2. Response with time of the count rate at two positions along the

cut papyrus stem at 15 cm (-) and at 65 cm (---) from cut end.Application time and tracer: (D) 20 s I32Pphosphate (2 !tCi/ml); () 20 s1' SCd"jEDTA2- (I itCi/ml). Deionized H20 was used between successiveapplications.

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956 VAN DE GEIJN

phase) is shown in Figure 4 for different Cd(N03)2 concentrationsand at three positions along the stem. The propagation speed ofthe tracer front, indicated by the time lag between first detectionat successive points along the stem (Fig. 4), changed almostlinearly with the concentration: After this non-steady-state phasethe count rate in all cases increased only at a low and constantrate (Fig. 5). At a fixed specific activity (jCi/4sg Cd), the countrate at the beginning of this steady-state was almost independentof the stable Cd concentration. A shift to deionized H20 causeda small and rather quick decrease in count rate followed by a

leveling. The amount of tracer decreased rapidly upon a shift toa non-labeled Cd(N03)2 solution (10-2 M), and finally tended tolevel again (Fig. 5). The transient peak, occurring immediatelyafter the switch to the 10-2 M Cd(N03)2 solution was due to thesudden change in ionic strength of the solution. Following theswitch the transpiration dropped to 10 to 20%1o of the initial rate

count rmte (cpm)Cd - EDTA deoized WotW

800

600

400

0 5 10 15 20 25 30 35time (min)

FIG. 3. Response with time of count rate for a 20-min application of a[15ICdmJEDTA - solution at 15 cm ( ), and at 65 cm (---) from cutend. Curves have not been corrected for differences in detector efficiencyand geometry.

count rate(cpmxxl13)

401

30

201

101

AND PETIT

count rate(cpm,

Plant Physiol. Vol. 64, 1979

0 2 4 6 8 10 12

time (min)

FIG. 4. Count rate response for different concentrations at three posi-tions: (i) at 15 cm; (X) at 40 cm; and 0 at 65 cm from the cut end.Cadmium nitrate concentrations: 10-' M; 2 X 10-3 M; (D 10-2 M.

Vessel cross-sectional area and transpiration rate were as indicated.

0 1 2 3 4 5 6 7 8 9 10 16

time ( h)

FIG. 5. Time course of the count rate at 15 cm (. ) and at 65 cm (--- -) in a typical experiment to determine the CEC. Sequence of treatments:(2) loading with 2 x 10-3 M [1"'Cdm]Cd(NO3)2 until steady-state; (2) wash with deionized H20; G) unloading with 10-2 M Cd(NO3)2 until steady-state;(i) wash with deionized H20; ( filling of the vessels with 10-2 M Na2EDTA + 10-4 M [ll5CdmlCdCl2; (0 wash with deionized H20. Count rate changesAC1 and AC2 are used in the equation to calculate the CEC.

A..

.'~~

-~~~~~~~~~~~~~~~~~~~~~~

*- ...I I

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CATION EXCHANGE CAPACITY OF XYLEM VESSELS

because of the large amount of salts imported into the leaves andthe consequent increase in osmotic pressure, causing a (partial)closure of the stomates. The drop in transpiration is reflected inthe delay in response of the second detector after the applicationof a [llSCdmJEDTA solution (Fig. 5).

Application of the [ll5Cdm]EDTA solution resulted in a fillingof the vessels with a solution of a known tracer concentration(uCi/ml), as described in the preceding section, and allowed thedetermination of the relative detector efficiency for the highenergy fl-rays of l5Cdm (19). Application should last sufficientlylong to prevent an interference with the residual llICdm removedby the excess EDTA, and to establish an equilibrium in the vessels.The CEC of the transport system per unit of vessel wall surface

now can be calculated from the equation (19):

AC1 ___ [*Cd-EDTAJ x 12xO3 eq CM-2 (1)CEC [Cd2&+] x C x2

xI*UE+]x 2 x

where [Cd2+] is the stable Cd concentration (M) during loading;AC1, AC2 is the difference in count rate, respectively, due tounloading and application of [l15Cdm]EDTA (cpm, Fig. 5);Bi irri, D 2iri is the total cross-sectional area (cm=) and circum-ference (cm) of the xylem vessels; and [*Cd2+], [*Cd-EDTAJ isthe concentration of l15Cdm (uCi/ml) in the solutions duringloading and CdEDTA treatment. The values obtained for theCEC in the various experiments are shown in Figure 6. The CECwas constant within the limits of experimental accuracy (about ±20%) over the Cd concentration range used here (10--10-2 M).Unloading of the stem with stable cadmium or Cd(NO3)2Ca(N03)2 (10-2 M) or Na2EDTA (10-2 M) did not influence theresults quantitatively. The kinetics, however, differed appreciably,Na2EDTA yielding the most rapid unloading. With the lattertreatment, the major part of the Cd from the exchange sites wasobviously chelated and rapidly transported in the bulk solution tothe top of the shoot (19).CEC Determinatio, Perfusion though Stem Segnents. The

tracer content ll5Cdm in successive perfused volumes of solutionin a typical example of a perfusion experiment is shown in Figure7. Cd has been used throughout this experiment. It is clear thatdeionized H20 can remove only in the transition period a smallamount of l15Cdm from the vessels or the exchange phase, asappeared also from the count rate measurements (Fig. 5). Theamount of Cd or Ca released upon a switch to Na2EDTA or astable Cd or Ca solution gives a close estimate of the CEC of thetransport system in the stem segment as shown in a previous paper

(Kfxmo(Cd orC 7cm2)

lo-

5

1l- Io.3 1o-2concentration Ca or Cd (M)

FIG. 6. Cation exchange capacity (mol Cd2+ or Ca2"/cm2 vessel wall)for various solution concentrations. Results from three procedures are

included. Each point represents a different plant stem. (0): Count rate

measurements (Cd); (0): perfusion experiments (Cd); (A): perfusion ex-

periments (Ca).

20 30vodume perfused (ni)

FIG. 7. Per cent recovery ofapplied activity concentration in successive5rfbsed volumes. Successive treatments: 05 x 1O-3M [115CdmlCd(N03)2;2) deionized H20; ® 10-2M Na2EDTA; [ll5CdmIEDTA; ® deionizedH20.

(19). Results from experiments with 45Ca and ll5Cdm are includedin Figure 6, and are in good agreement with the values calculatedfrom the count rate measurements.

DISCUSSION AND CONCLUSIONS

The existence of a fixed CEC in the xylem transport system hasbeen indicated by: (a) the measured linear relationship betweenthe translocation speed and the Cd24 concentration (Fig. 4); and(b) the absence ofa concentration dependence ofthe tracer contentat the start of the steady-state phase observed after loading withCd at a fixed specific activity. These two phenomena can indeedbe explained considering the xylem as an exchange column witha fixed number ofexchange sites per unit of length. Translocationshould, in this concept, proceed by shifting the labeled cationsalong the exchange column at a rate depending on solutionconcentration and transpiration. In such a column virtually allexchange sites would have to be saturated, irrespective of thecation concentration in the supplied solution. This is true, as canbe seen from the relative inability of water to remove Cd or Cafrom the exchange sites (Fig. 5) and also from the absence of adependence of the CEC on cation concentration. The binding ofthe cations (Ca24 or Cd2+) by the exchange sites is thus verystrong.The affinity of EDTA for Cd (log k1 = 16.6) and Ca (log k1 =

10.7) (17) apparently is sufficient to compete effectively with theexchange sites: Na2EDTA removes these cations from the sites,and both the CdEDTA and CaEDTA anionic complexes aretransported with the transpiration flux, comparable with 3P-la-beled phosphate.These observations are in full agreement with results reported

by other authors. The exchange type of transport of Ca wasdemonstrated first by Biddulph et al. (2), a principle confirmedand extended by many others (5, 8, 9, 16). Ca was shown to beexchangeable with other divalent cations (1, 5, 16), a processwhich appeared to take place also between Cd2+ and Ca2+ instems of intact tomato plants, where Ca replaced Cd in a sequenceof Cd loading/unloading periods (12). An increase in mobility ofCa in the presence of EDTA has also been reported (4, 8). Thepresence of chelating agents will be effective only if the stabilityof the metal-chelate complex is sufficient to compete with theafffmity of the exchange sites for the metal cation (5). Measure-ment ofCa complexes in collected xylem exudate is, therefore, byno means a proof for the existence of these complexes in thetransport channels (3).The charge density required to explain the high value of the

CEC (3-5 x 108 mol Ca2+ or Cd24/cm2 vessel wall) is too high(3.6-6 charges per A) to assume it to be located on the surface

0 9~~~00 ~~~~0 a

o o ^ o~~~~

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VAN DE GEIJN AND PETIT

only, e.g. the inner vessel wall. The binding of Ca2" and Cd2"(ionic radii 0.99 and 0.97 A, respectively) would require a multi-layer situation. The CEC, as determined here, should, therefore,be interpreted as the cation exchange capacity associated with 1

cm2 surface area of vessel wall. Klein and Ginzburg (10) showedthat cations (Ca, Mg, Cu, Fe, Mn and Zn) have cementingproperties in cell walls. They also demonstrated that EDTA mayremove these metals, and thereby loosen the laminar structure ofthe cell wall, a process which was reversible. The possible locationof the charge sites inside this laminar structure consisting of up toa 100 layers (14) could account for a 100-fold reduction of thesurface charge density, bringing it down to a more realistic value:0.6 to 1.0 x 10-9 eq cm-2, or 3.6 to 6 charges/100 A2-layer.

Alternatively, assuming the vessel walls to have a thickness of2 ,um, 0.6 to 1.0 X 10-7 eq cm-2 corresponds to a charge concen-tration of 300 to 500 mm, which is in excellent agreement withPitman's (13) estimation of the density of charges in the Donnanphase of cell walls (300-600 mM).As no directly comparable measurements are available, our

measured CEC was recalculated on a fresh weight basis. Ourvalues (2-3 x 10-6 eq/g fresh weight) are in accordance with thosereported by Fried et al. (6) determined from absorption experi-ments with intact barley roots (4 x 10-6 eq/g fresh weight for Sr).Their and our values are lower by about a factor of 10 comparedto the CEC determined by titration and isotopic exchange methodswith roots (4), stem, and leaf tissues (11). From the present resultsit must be concluded that part of the exchange sites determinedby titration methods do not participate in the Ca transport proc-esses in the xylem.The results reported here are of general interest as they are

obtained with an intact shoot exhibiting a normal upward trans-port, and are in accordance with phenomena observed with intactplants (12). The relative simplicity of the approach using semicon-ductor detectors and high energy ,8-emitters (ll5Cdm, ESP) allowsfurther investigations in the field of cation transport, their inter-actions and selectivity coefficients for the exchange sites, and theinfluence of exogeneous and endogeneous chelators on the trans-port.

Plant Physiol. Vol. 64, 1979

Acknowledgment-The skillful assistance of H. Roelofsen was indispensable. The intercstingand carifying discussions with many colkagues, especially with Dr. M. J. Frissel and Dr. F. van

Dorp are gratefillly acknowledged.

LITERATURE CITED

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2. BIDDULPH 0, FS NAKAYAMA, R CORY 1961 Transpiration stream and the ascension ofcalcium.Plant Physiol 36: 429-436

3. BRADFIEL EG 1976 Calcium complexes in the xylem sap of apple shoots. Plant Soil 44: 495-499

4. CRooKEWM 1964 The measurement of the cation-exchange capacity of plant roots. Plant Soil21: 43-49

5. FERGUSON IB, EG BOLLARD 1976 The movement of calcium in woody stems. Ann Bot 40:1057-1065

6. FRiED M, JC NoGGLE, CE HAGEN 1958 The relationship between adsorption and absorptionof cations. Soil Sci Soc Am Proc 22: 495-499

7. HEINE RW 1970 Absorption of phosphate and potassium ions in poplar stems. J Exp Bot 21:497-503

8. IsERMANN K 1978 Einfluss von Chelatoren auf die Calcium-Verlagerung im Spross hohererPflanzen. Z Pflanzenern Bodenk 141: 285-298

9. JAcoBY B 1967 The effect of roots on calcium ascent in bean stems. Ann Bot 31: 725-73110. KLmwN S, B GINZBURG 1960 An electron microscopic investigation into the effect of EDTA on

plant cel wall. J Biophys Biochemic Cytol 7: 335-3381 1. KNIGHT AH, WM CROOKE, RHE INKSON 1961 Cation-exchange capacity of tissues of higher

and lower plants and their related uronic acid contents. Nature 191: 142-14312. PEsTr CM, SC VAN DE GEUN 1978 In vivo measurement of cadmium ("5mCd) transport and

accumulation in the stems of intact tomato plants (Lycopersicon esculentum Mill). I. Longdistance transport and local accumulation. Planta 138: 137-143

13. PImTAN MG 1977 Ion transport into the xylem. Annu Rev Plant Physiol 28: 71-8814. PRESTON RD 1974 The Physical Biology of Plant Cell Walls. Chapman and Hall, London15. RiNGoET A, RV RECHENMANN, H VEEN 1967 Calcium movement in oat leaves measured by

semiconductor deectors. Rad Bot 7: 81-9016. SHEAR CB, M FAUST 1970 Calcium transport in apple trees. Plant Physiol 45: 670-67417. SuILN LG, AE MARTEL 1964 Stability constants of metal-ion complexes. Sp Publ 17, Ed 2,

Chemical Society, London18. VAN DE GEuN SC 1973 Some considerations conceming microlocalization in dehydrated and

living plant material. II. Beta spectrometric localization in living plants. Trans 3rd SympAccumulation and Translocation ofNutrients and Regulators in Plant Organisms. Jablonna,Skierniewice, Brzezna, Krak6w, Proc Res Inst PomoL Sckiemiewice, Warsaw, Poland, SerE No. 3, pp 579-587

19. VAN DE GEuN SC, CM PETIT, H RoELOFSEN 1979 Measurement of the cation exchangecapacity of the transport system in intact plant stems. Methodology and preliminary results.Comm Soil Sci Plant Anal 10: 225-236

20. WACQUANT JP, L PASSAMA 1971 Sur les relations entre l'adsorption radicellaire preferentielleet absorption selective de Ca, Mg, K et Na chez diverses populations dAnagallis arvensis L.et d'autres especes. CR Acad Sci Paris 272 D: 711-714

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