OF - · PDF file100 * by flame photometry. The response in terms of K 115 8 absorption paralleled that in the present experiment 215 1 on absorption of Rb. Is calcium the only divalent

THE ESSENTIAL ROLE OF CALCIUM IN SELECTIVE CATIONTRANSPORT BY PLANT CELLS 1

EMANUEL EPSTEINDEPARTMENT OF SOILS & PLANT NUTRITION, UNIVERSITY OF CALIFORNIA, DAVIS

The mechanisms whereby plant cells absorb in-organic ions from the external media are selective:they discriminate among different ions. Particularlyintriguing is the discrimination between potassiumand sodium ions. Most plant cells accumulate muchhigher concentrations of potassium than of sodiumions, even from media in which the sodium concentra-tion greatly exceeds that of potassium.

Perhaps the best way to investigate these specifici-ties is to study the manner in which different ionsaffect each other's absorption. In a previous paper(4) experiments were described demonstrating thatin excised barley roots potassium and rubidium com-pete with each other in an identical absorption mech-anism. At moderate concentrations, neither sodiumnor lithium ions competed for the potassium-rubidiumtransport mechanism. The deduction was that thereare specific ion binding sites on carrier moleculeswhich effect the transport of the ions across cellularmembranes. Potassium and rubidium ions are boundand transported by sites which fail to discriminatebetween them, hence these ions compete with eachother for identical carrier sites. Sodium and lithiumare not bound by the potassium-rubidium sites, hencedo not compete with potassium and rubidium but aretransported by separate sites.

In this paper experiments with barley roots arepresented which show that calcium ions are essentialfor the integrity of the selective absorption mechanismreferred to, and information is given about some quan-titative features of the calcium effect.

MATERIALS & METHODSThe distilled water piped into the laboratory was

passed through a mixed ion exchange column2 tofree it from heavy metal and other impurities. Thispurified water was stored in pyrex carboys. Glass-ware was rinsed with about 0.5 M HCl, followed byexhaustive rinsing with distilled water. Salts used

'Received January 9, 1961.2 Bantam Barnstead Demineralizer, made by Barnstead

Still and Sterilizer Co., Boston 31, Mass.

were C.P. grade. Radioactive Rb86 and K42 wereobtained from Oak Ridge National Laboratory, andNa22 from Nuclear Science & Engineering Corp.,Pittsburgh 36, Pa. The stock solutions were labeledto the extent of about 0.002 uc per /Amole.

PREPARATION OF ROOT MATERIAL. Seeds of bar-ley, Hordeum vulgare L. var. Arivat, were allowedto germinate for 24 hours in 1 liter aerated water inthe dark, at room temperature (about 200 C). Thewater was renewed once during this time, 19 hoursafter the start of the germination period. The ger-minating seeds were spread on a layer of cheeseclothwhich had been washed and rinsed with distilledwater. The cheesecloth was supported by a stainlesssteel screen about one centimeter above the surfaceof 4 liters of 2 X 10-4 M CaSO4 solution in a 4-literpyrex beaker. A second cheesecloth was spread overthe seeds. The corners of both cheesecloths dippedinto the solution. An inverted watchglass, 17.5 cmin diameter, was placed on the beaker. The topcheesecloth was removed as the germinating seedlingspushed it up.

This assembly was placed in the dark, at roomtemperature. The solution was aerated continuously.Three days after the seeds were planted on the cheese-cloth, the stainless steel screen with the seedlingswas removed from the beaker, the roots were rinsedin two changes of water, and the assembly put on abeaker with fresh solution. The watchglass was notreplaced. Two days after this, the roots were rinsedrepeatedly in water, excised just below the screen,rinsed several more times, and suspended in aboutfour liters of water.

ABSORPTION EXPERIMENTS. For the experimentsproper, roots were gently blotted on dry cheeseclothwhich had been washed and rinsed with distilledwater. One-gram samples were weighed out on atorsion balance and transferred to 100 ml water in30 X 300 mm culture tubes. The tubes were placedin a waterbath maintained at 30° C, and the water inthe tubes was aerated. To start the absorption periodproper, each tube in turn was removed from the bath,the water was decanted, the roots were rinsed twicewith about 100 ml water each time, and 100 ml ofprepared experimental solution were then poured in.The tubes were replaced in the bath, and the solutionwas kept aerated.

437

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PLANT PHYSIOLOGY

The absorption period w-as discontinued by decant-ing the experimental solution and rinsing the rootsfor 1 minute with a total of at least 500 ml water.

All salts of the experimental solutions werechlorides. The initial pH (unbuffered) was 4.9, andthis changed by no more than 0.2 pH units duringthe experimental runs. The roots contained initiallyno detectable Rb, less than 1 ,umole Na per gramfresh weight, and about 15 ,umcvalues varied slightly from experiralthough all seed used came from

RADIOACTIVE ASSAY. The rtransferred to 1-inch X 5/16-inchchets, dried, and ashed at 5000 C.one drop detergent solution were athe samples dried under infr-samples were counted with a thincm2) Geiger-Mueller tube connectescaler. Each sample was countec3,000 counts each time. In most e)were counted to 10,000 counts ea

of an appropriate dilution of the radtion were pipetted into counting ct

tion of a drop of detergent were drlamps.

FLAME PHOTOMETRIC ASSAY.were transferred to 30-ml pyrex tashed at 5000 C. The ash was tHNO3, and assayedl in a Beckman Iphotometer with photomultiplierment. Standard curves were prelsolutions of the salts in 0.16 N I

was assayed at 766 mu, so(lium atiuni at 780 m,u.

TABLE IEFFECTIVENESS OF DIVALENT CATIO

INHIBITION OF Rb ABSORPTION

0

0

202020

202020202020

. . .

Ca

MgCa

SrBaMnCoNiZn

Rb ABSORi

tmole/g/hr

6.66.92.02.34.3

3.23.54.02.82.63.7

CONSISTENCY & RELIABILITY OF RESULTS. Foreach experiment reported, one or several others weredone with or without modifications. No results wereobtained that were inconsistent with those reported.

RESULTS

)le K. These two The effect of Na on absorption of Rb during anent to experiment, 60-minute period is shown in figure 1F. In the ab-the same batch. sence of Ca, Rb absorption from a 1 mi1v solution of

RbCl was progressively diminished by increasing con-

ot. samples were centrations of NaCl. In the presence of Ca, the effectA lickl-pated and of Na was quite different, andl this typical response

to Na was showni in experiments both on Rb anid K(lde(le to the ashand absorption (figs 1, 2, 3, 5). There always is a slightt-red lamps. inhibition of absorption at 1 to 2 mM\ Na. Between 5

-windowv(1.4 mg and 10 mat Na, absorption of K or Rb rises to almostd to.a conventional the control (no Na) value, an(l at still higher Na

l twice, to at least concentrations declines slightly.

pertimen. Samples This marked indifference of K andl Rb absorptionlch time. Samples to even high concentrations of Na. in the presence ofxlioactive stock solu-

Ca, is in coInspicuous contrast to the pronounced in-ips and after addi- hibition of K absorption by Rb (fig 2). an(d of Rhieel under infra-red absorption by K (fig 3). Both of these experiments

lasted 60 minutes. and the K an(d RI) concentrationThe root samples was 1 mr. Calcium w\vas present at a concentration

beakers, dried, and of 0.05 mar.Laken up in 0.16 N In the previous experiments, the concentrationMlodel DU spectro- of Ca was 0.05 nmar. The effect of varying the Caand flame attach- concentration over a w\ide range is slhown in figurepared from known 4. The Rb concentration was 1 mar and that of NaINO.. Potassium 20 mna throughout. Calcium concentrations are plot-589 ms,u and rubid- ted con a logarithmiiic scale, experimental points lying

at full poxvers of ten and three times these values. Atthe Na concentration used (20 mar), RI absorptionfroml a 1 mat solution was reducedl to 2.1 tsmole/g/hour. in the absence of Ca, anld there was no effectfrom Ca at 1 X 10-6 N. At higher Ca concentra-

BY Na IONS*tions Ca became increasingly effective in reversingthe inhibition due to Na, Rb absorption rising moststeeply at about 1 X 10-4 N Ca, and leveling off at

°TION RANK OF 3 X 10-4 to 1 X 10-3 N Ca. In a similar experiment

% OF DIVALENT with K as the ion absorbed, the Na concentration was

)NTROL** CATION 10 man, and the absorption of K from 5 mt solution

...... rwas (letermined as a function of the Ca concentrationover the same range. Assay in this experiment was

100 * by flame photometry. The response in terms of K115 8 absorption paralleled that in the present experiment215 1 on absorption of Rb.

Is calcium the only divalent cation capable of pro-160 5 tecting the K-Rb absorption meclhanism against the

175 4 Na inihibition? Table I gixes the results of an ex-200 2 periment in whiclh the effectiveness of eight (livalent140 6 cations was teste(l. The first two lines show results

130 7 obtained without Na in the solution. In the absence185 3

Asterisks in the figureS ',ii- te radioactively lab) ledions.

Na CONC, DIVALENTMM CATION

* Conicentration of Rb: 1 mat. Concentration ofdivalent cations: 0.05 mar; time: 60 minutes.

** The sample with Na present at 20 mm concentra-tion, but without added divalent cation, is considered thecontrol (100 74 )

438



EPSTEIN-CALCIUM ROLE IN CATION TRANSPORT

of Na, Ca at 0.05 mm does not appreciably increasethe amount of Rb absorbed. The severe inhibitionof Rb absorption by Na (line 3) is reversed by Ca(line 5) and the other divalent cations tested, to vary-ing degrees. Calcium is the most effective ion. Thelast column of table I ranks all ions tested in order oftheir effectiveness.

A curious result (table I) was the ineffectivenessof the Mg ion in affording protection against the Nainhibition of Rb absorption. The value to which theNa inhibition reduced Rb absorption, viz. 2.0 Amole,was more than doubled in the presence of Ca, but Mgat the same concentration increased it by merely15 %. No other ion, among those tested, was equallyineffective. Figure 5 shows the results of an experi-ment in which both Ca and Mg were studied in theirrelative effectiveness in counteracting the Na inhibi-

tion of K absorption. The roots absorbed from 1 mMsolutions of K, for 60 minutes. At the lowest Naconcentrations Mg had some effect, but at the higherconcentrations of Na the effectiveness of Mg declinedand at 20 mm Na, K absorption in the presence ofMg was not significantly higher than in absence ofany divalent cation. Calcium largely protectedagainst Na inhibition, as usual.

The effect of time was studied in an experimentin which roots absorbed Rb from a 1 mM solution inthe presence, throughout, of 20 mm Na. In one set,Ca was present at 0.1 mm from the beginning.Another set had no calcium. A third was withoutCa for the first 24 minutes of the absorption period,then Ca was added in 0.1 mm ooncentration. Theresults are shown in figure 6.

The foremost features in this experiment are as

No or Rb, mM

10 20 - lb 110' 1a 4lo, 03No or K, mM Co, N

FIG. 1. Effect of Na on absorption of Rb in the absence and presence of Ca. Rb: 1 mM; time: 60 minutes.FIG. 2. Effect of Na and of Rb on absorption of K in the presence of 0.05 mm Ca throughout. K: 1 mM; time:

60 minutes.FIG. 3. Effect of Na and of K on absorption of Rb in the presence of 0.05 mM Ca throughout. Rb: 1 mM;

time: 60 minutes.FIG. 4. Effect of Ca concentration in reversing the inhibition of Rb absorption by Na. Rb: 1 mm; Na: 20

mm-; time: 60 minutes.

439



PLANT PHYSIOLOGY

follows: In the presence of Ca, Rb absorption wasa strictly linear function of time, extrapolating to zeroat zero time. Without Ca, the rate of absorptiondeclined all along. Upon addition of Ca to the solu-tions lacking Ca there was no immediate effect onthe rate of Rb absorption, but after about an 8-minutelag the rate of Rb absorption increased. The re-covery was not complete, however, in that these rootsdid not absorb Rb as fast as did those that had Capresent from the beginning of the absorption period,nor was the rate of absorption constant.

In the previous experiments it has been shownthat in the presence of Ca at concentrations of 0.05to 0.1 mm, Na over a wide range of concentrationsfails to compete with either K or Rb in the absorptionprocess (figs 1, 2, 3, 5), while K and Rb do mutuallycompete (figs 2, 3). What is the converse effect,that of K or Rb upon the absorption of Na? Figure

10

X 8 M 0.05 mM

¢7Co ~~~~~~~~~CONTROL

(I |I Iv0 5 10

No, mM15 20

7 shows the results of an experiment in which Naabsorption from a 1 mM solution was studied as af-fected by increasing concentrations of K, in the ab-sence and presence of Ca at 0.1 mm concentration.The absorption period was 60 minutes.

In the absence of Ca, Na absorption progressivelydeclines with increasing K concentration, much asRb absorption and K absorption are decreased byprogressively higher concentrations of Na, withoutadded Ca (figs 1 & 5). In the presence of Ca, on theother hand, there is a sharp drop in Na absorptionat 1 mM K. At higher K concentrations, Na uptakedecreases much more gradually, and virtually levelsoff at the highest K concentrations tested. Thesefeatures are brought out even more clearly in the ex-periment shown in figure 8, identical with the previousone in every respect except that the Na concentrationwas 5 mM.

6

4

53

aiLonD--r 4

+Ca

-Go - +CaX

Ca added to -Ca cultures

50 60 700L 10 20 30 40MIN.

x 8I

-3

_i 6

CD4co

=

2

0

Ca, 0.1 mM

10 15 20K, mM K, mM

FIG. 5. Effectiveness of Ca and Mg in reversing the inhibition of K absorption by Na. K: 1 mM; time: 60minutes.

FIG. 6. Time course of Rb absorption in the presence of Na, and the effect of Ca. Rb: 1 mM; Na: 20 mM;Ca where present: 0.1 mM.

FIG. 7. Effect of K on absorption of Na in the absence and presence of Ca. Na: 1 mIm; time: 60 minutes.FIG. 8. Effect of K on absorption of Na in the absence and presence of Ca. Na: 5 mM; time: 60 minutes.

ui 6.-jC>mZi 5ci,L"4m 4m10cnm 3:1

n Kt

0 I

440

101

5




TABLE IIABSORPTION OF Rb & Na AS A FUNCTION OF NaCONCENTRATION IN ABSENCE & PRESENCE OF Ca*

Na CONC Rb ABSORBED, ,umole Na ABSORBED, Amolemm --Ca +Ca -Ca + Ca

0 6.5 6.6 ...

5 5.0 6.4 5.4 3.910 4.3 6.6 8.7 5.720 3.5 6.8 13.5 8.0

* Concentration of Rb: 1 mm throughout. Calcium,where present: 0.1 mM. Absorption period: 60 minutes.Experiment done in duplicate, one set with Rb* for radio-active assay, the other with Rb for Na assay.

In table II the results of an experiment are pre-sented in which the absorption of Rb and Na wasmeasured both in the absence and presence of Ca at0.1 mAi. The Rb concentration was 1 mm; Na rangedfrom zero to 20 mm. In stabilizing the K-Rb ab-sorption mechanism against inhibition by Na, Caappreciably reduces the absorption of Na (cf. figs7 & 8).

DISCUSSION

The results confirm and extend previous conclu-sions (4): K and Rb ions are mutually competitive,Na does not compete in at least one K-Rb absorptionmechanism. In the present work Ca is shown to beessential for the maintenance of the integrity of thisselective ion transport mechanism.

In particular, Ca prevents Na from competing withK and Rb at the higher Na concentrations. In theearlier experiments, Na at low concentration (up to10 nim) did not compete with Rb, but it did competeat higher concentrations. It is shown here that with-out Ca, the selectivity between K and Rb on the onehand and Na on the other breaks down even at lowconcentrations of Na. Evidently enough endogenousCa was present in the earlier experiments to permitdemonstration of the relative indifference of the K-Rbtransport mechanism to Na. In the present experi-ments another variety of barley was used, the volumesof experimental solution were larger, and the rootswere much more exhaustively rinsed before the ab-sorption period. These factors made for a morethorough removal of diffusible Ca from the tissue,and permitted demonstration of the essentiality of Cain the selective ion transport mechanism.

In analyzing the results, a convention adoptedearlier (4) will be used. The ion whose absorptionis discussed at the moment will be called the substrateion, and the ion whose effect on the absorption of thesubstrate ion is being tested will be the interferingion. As a matter of fact, both the substrate and theinterfering ions are absorbed, and the terminologyis used for convenience only.

The results shown (figs 2 & 3) are consistentwith the previous interpretation (4) that K andRb are transported by identical carrier sites and com-pete with each other for these sites. With K as thesubstrate ion, Rb at increasing concentrations pro-gressively diminishes the absorption of K indicatingthe progressive displacement of K ions by Rb ionsfrom the available carrier sites (fig 2). Competitionfor identical sites is mutual, by definition, and figure3 shows that K in quite similar manner reduces theabsorption of Rb. The identity of the K and Rbtransport mechanisms is further brought out by thefact that the absorption of both is affected in similarmanner by Na ions, in the presence of Ca (figs 1.2, 3, 5). One cannot tell, without reading the leg-ends, whether a given graph depicts the effect of Naon K or Rb absorption.

The K-Rb carrier mechanism is highly indifferentto even a 20-fold excess of sodium in the solution,but only in the presence of Ca (figs 1, 5). Evidentlyunder these conditions Na, over a wide range of con-centrations, fails to compete for the K-Rb carrier sites.This difference between Na on the one hand, andK-Rb on the other, is strictly a phenomenon of cationselectivity, distinct from any anion effect. The con-centration of Cl ions accompanying the interferingcation increased progressively in precisely the samemanner, whether Rb or Na was the interfering cation(fig 2). The sharp contrast between the effects ofthese two chlorides on K absorption was therefore dueentirely to the respective cations. The same con-siderations hold for the findings shown in figure 3.

Calcium has been shown by Viets (14) and othersto accelerate the absorption of various ions by planttissues, including the absorption of K (8, 11, 14) andRb (3) by barley roots, the absorption of Rb bymung bean roots (13) and the absorption of K bycorn and soybean roots (10). Is the Ca effect de-scribed in the present paper identical with this earliereffect of Ca and other divalent and trivalent cationson ion absorption? The answer to this question isno, for several reasons.

I. The effect described heretofore of acceleratedion absorption is produced not only by Ca ions butby several other divalent cations as well, includingMg ions. In all but one of Viets's (14) experimentsdealing with the comparison of various divalent ca-tions, Mg was second only to Ca in accelerating ionabsorption by barley roots. Tanada (13) also foundthat results obtained with Mg were essentially similarto those obtained with Ca in experiments on the in-fluence of these ions on the absorption of Rb andH,PO4 by mung bean roots. In the present experi-ments, Mg was the least effective among the divalentions tested (table I, fig 5).

II. The Ca effect described in the present paperapproaches a maximum at Ca concentrations on theorder of 3 X 10-4 to 1 X 10-3 N, and is about halfmaximal at 1 X 10-4 N Ca (fig 4). The acceleration

441



PLANT PHYSIOLOGY

of ion absorption described by Viets (14) and others(3, 11, 13) was caused by Ca at much higher concen-trations, saturating Ca concentrations being usuallyon the order of 1 X 10-2 N or even higher.

III. The previous reports referred to experimentsin which the experimlenital solutions contained twvosalts: the salt of the substrate ion wlhose absorptionwas stu(lied, and a salt of Ca or some other divalentor trivalent cation. Under similar con(litions, i.e.,in the absence of Na, Ca at concentrations of 0.05 mmor 0.1 nim did not accelerate the absorption of Rbsignificantly (table I, 1st 2 lines; table II, 1st line;figs 1, 5). Only when the absorption of K or Rbwas inhibited by Na (lid Ca at these concentrationsincrease their absorption.

It is conclude(d that the Ca effect (lescribe(l here(liffers from that discussedl by Viets an(d otlhers.Their experiments showed that dlivalent cations causedan acceleration of ion absorption per se. The presentexperiments dlescribe a reversal, by Ca and certainother (livalent cations, of the inhibition of K-Rbtransport by Na. For this reversal. Ca is effectiveat much lower concentrations than those requiredl forstraightforward acceleration of ion absorption, andMg is largely ineffective.

In the presence of Na at 20 mw the rate of Rbabsorption steadily decline(d if no Ca was present,but remained constant in the presence of 0.1 mM Ca(fig 6). The findings suggest a progressive derange-ment of the K-Rb transport meclhanism by Na, in theabsence of Ca. Recovery froml this Na- induced im-pairment uponl a(l(lition of Ca was neither immediatenor complete. It took more than 8 minutes afterad(lition of the Ca before the rate of Rb absorptionincreased, an(d even then it renmained lower and lesssteadly than the rate of absorption 1w those roots thathad Ca present from the beginning. The lag in therecovery after addition of Ca was slightly shorter inanother experiment similar to that shown in figure 6,being a little less than 7 minutes. Such a lag is notsurprisinig in v\iew of the fact that the initial penetra-tion of divalelnt cations ilnto the tissue is fairly slow,about 75 (4 equilibration being reached in 8iminutes(5).

Inasmiiuclh as Na, in the presence of Ca, failed tocompete with K-RI) over a wide range of concentra-tions, experiments were (lone to examine the recipro-cal effect, viz.. the influence of K-Rb on the absorp-tion of Na. Absorption of Na was (Iepressed by Kmore severely in the presence of Ca than in its ab-sence, but comparison is invitedl of this K effect onNa absorption (figs 7 & 8) w\ith the straightforwardcompetitive situation, as when K conmpetes with RI)(fig 3), or Rb witlh K (fig 2). The latter responsesare consistent witlh the previous interpretation (4)that these two ions, K andl Rb, (lisplace each otherfroml i(lentical carrier sites. As the ratio of interfer-ing to substrate ions is progressively raise(l, comlplete

(lisplacement of the substrate ions by the interferingones is asymptotically approached.

Clearly, such a simple competitive situation doesnot prevail when K interferes with the absorption ofNa, in the presence of Ca (figs 7 & 8). At low Kconcentrations there is a pronounced inhibition ofNa absorption, but at the higlher concentrations of Kthe response of Na absorption to added incremlentsof K becomes very slight, especially wlhen the Na con-centration is 5 mIi (fig 8). This is interprete(l asindicating the existence of at least two Na carriersites. The operation of one of these is inhibite(d byK, the other one is quite indifferent to K andl accountsfor the absorption of Na at the higlher concentrationsof K.

A douible nmechaniislmi is suggeste(l for K-RI) absorp-tion as well. Figure 2 shows that at low concentra-tions, Na interferedl somewhat with K absorption, in-(licatilng a Na-sensitive K tranisport nmechaniismii, butthe mlechlaniismii effecting K absorption at the higlherNa concentratioi)s was quite inclifferent to Na. Thesame phenomenon is apparent to a lesser extent inthe other experiments on Na interference writh K-Rbabsorption, in the presence of Ca. It is mlore pro-nounce(I when the K-Rb concentrationi is hiigher (see4, fig 1).

In these experimiienits it has been shown that fromlsolutions containing botlh K (or Rb ) and Na, thetissue absorbs relatively more K (or RI) and rela-tively less Na in the presenice of Ca tlhani in its absence.It might be tempting to a(lvance the expllanation thatCa mliinimizes Na interference witlh K-Rb transportby excluding Na, thus preempting carrier sites forK-Rb transport by preventing Na from having accessto theml. However, the facts (lo not support the i(leaof a simple one-to-one-relationship. Table II shows,as dlid the experiments presente(d in figures 1. 2, 3, and5, that Ca reversed the progressive inhibitioni of theK-Rb transport by ever larger concenltratioins of Na.Nevertlheless, absorption of Na, thouglh less than inthe absence of Ca, increasedl wvith increasing Na con-centrations even in the p)resence of Ca, an(d at thehighlest Na conceintration, 20 nm.r, exceedled the ab-sorption of Rb. At this concentration Na, in theabsence of Ca, caused a drop in Rb uptake of 3 ,unLoles.from 6.5 to 3.5 ,umoles, wlhile 13.5 iumoles Na wereabsorbed.. In the presence of Ca, Rb absorption wasessentially unaffected, ancl 8.0 uamoles Na were ab-sorbed at the highest Na concentration.

The following hypothesis is consistent with thefacts. Two species of carrier sites transport K-Rb.One of these, A, with a relatively low affinity forK-Rb ions, is inhlibitedl by Na (fig 2), an(l miioreseverely so, the higher the K-Rb concentration (4,fig 1). This latter feature is (lue to the fact that thislow-affinity site accounts for a greater proportionof the total K-Rb transport at the higher K-Rb con-centrations. The inhibition by Na of K-Rb transportby this site is mitigated but not completelv reversedby Ca (figs 1, 5). The other site, B, has a higheraffinity foi- K andl, in the presence but not in the ab-

442




sence of Ca, is largely unaffected by Na. This is thesite responsible for the lack of inhibition of K-Rbtransport at the higher Na concentrations when Cais present (figs 1,2, 3, 5).

Two carrier sites are indicated for the transportof Na as well. In the absence of Ca, Na is trans-ported by a site, C, with relatively low affinity forthe ion. When Ca is present this site is largely pre-empted by K (figs 7, 8, over the lower range of Kconcentrations). Another part of the Na transportis through a site, D, with high affinity for Na ionsand largely indifferent to K, in the presence of Ca(figs 7, 8, at the higher concentrations of K).A careful consideration of all the data suggests

that the K-Rb transporting site A may be identicalwith the Na transporting site C, and to that extentthere exists indeed a mechanism common to K-Rband Na transport. This site has a much higher af-finity for K than for Na when Ca is present, hencethe sharp drop in Na absorption at even low K con-centrations, in the presence of Ca (figs 7, 8).

The significance of these findings lies in the factthat plant cells under normal physiological conditionsabsorb K and Na in the presence of Ca. Only thepresence of sufficient Ca of endogenous origin per-mitted the earlier demonstration (4) of distincttransport sites for K-Rb and Na, respectively. Whenthe precautions described here are taken, this selec-tivity breaks down if Ca is omitted from the solutions.

This conclusion probably applies to plant cellsgenerally. The red alga, Porphyra perforata, differsin its K and Na transport from barley roots in thatit accumulates K and extrudes Na, but here, too. Cais essential in maintaining the integrity of the distinctK and Na transport mechanisms (2). Jacobson et al(9) have shown that the roots of plants of severalspecies absorb more K and less Na in the presence ofCa than in its absence. A general impairment of iontransport through removal of Ca has been describedby Hanson (6) and Helder (7), but their studies didnot dleal with the selective feature of K-Rb vs. Natransport described here.

Certain other divalent cations besides Ca, notablyMn, can partially reverse the inhibition of K-Rbabsorption by Na (table I), yet Ca probably is the ionchiefly responsible for the maintenance of ion selec-tivity under normal, physiological conditions. Phys-iological concentrations of such divalent micronu-trient cations as Mn and Zn are on the order ofmicromoles per liter, but in this range of concentra-tions even Ca was without much effect (fig 4). Man-ganese and zinc, when tested at a concentration of5 wi (1 X 10- N), were about as effective as Caat that concentration (cf. fig 4). Only Mg, amongthe divalent cations tested, is present in natural sub-strates at concentrations of the same order of magni-tude as Ca ions, and it was quite ineffective in revers-ing the inhibition, by Na, of K-Rb absorption (tableI, fig 5).

The present findings strengthen the earlier con-clusion (4) that in barley roots and probably quite

generally in plant cells, there are carrier sites involvedin the transport of K-Rb which are distinct fromothers that transport Na ions. The opposite view,that there is a single common mechanism responsiblefor the absorption of these cations, has been discussedin the literature (1, 9, 12). Particularly relevant arethe conclusions of Conway and Duggan (1) on ionabsorption by yeast because they used the same kinetictreatment (4) that had led to the finding of separatemechanisms of K and Na absorption in barley roots.They concluded that in yeast, absorption of K andNa is by one and the same carrier mechanism. Theseexperiments with yeast were done at high concentra-tions of the monovalent cations (up to 100 mm Na),in the absence of divalent cations. It is thus quitepossible that different results would have been ob-tained under more nearly physiological conditions,which include the presence of Ca or other divalentcations.

The present results cannot be explained by ascheme of simple mutual competition by K-Rb andNa for a single common carrier site. The operationof at least three sites is demonstrated of which oneis highly selective for K-Rb and another for Na. Thepresent findings confirm the validity of the "enzymekinetic" analysis of absorption rates which initiallyled to the conclusion of separate K-Rb and Na trans-port sites (4).

SUMMARYExcised barley roots, rinsed thoroughly free of

diffusible Ca, absorbed ions from solutions containingKCl or RbCl or NaCl, or combinations of these, inthe presence or absence of Ca or other divalentcations, for periods of 60 minutes in most experiments.

In the absence of Ca, Na interferes with K ab-sorption, and K with Na absorption, at all concentra-tion ratios examined. In the presence of Ca, Na in-terferes slightly with K absorption at low Na con-centrations (1-2 mM), but at progressively higherconcentrations of Na there is little or no further in-hibition of K absorption.

In the presence of Ca, K inhibits absorption of Naat low K concentrations, but as the K concentrationsare raised inhibition of Na absorption per added in-crement of K becomes very slight.

Potassium and rubidium are mutually competitivein the presence of Ca.

Calcium is maximally effective in maintainingthe integrity of the selective absorption mechanismsat a concentration of about 1 X 10-3 N. Other di-valent cations, notably Mn, are also active, but Mgis largely ineffective.

When Ca is added to solutions from which rootsabsorb Rb in the presence of Na, the impairment ofRb absorption by the Na present is only partiallyreversed, and only after a time lag of some minutes.

It is concluded that transport of K-Rb is throughtwo carrier sites, one inhibited by Na, the other large-ly indifferent to this ion. Transport of Na also in-

443



PLANT PHYSIOLOGY

volves two carrier sites, one of which is largely in-different to K-Rb. Calcium is essential to the in-tegrity of the selective ion transport mechanisms.

ACKNOWLEDGAT ENTSI want to thank Dr. Roy Overstreet for making

available to me a quantity of Na22. The barley seedwas obtained from the Agronomy Department,through the courtesy of Dr. Charles W. Schaller.

LITERATURE CITED1. CONWAAY, F. J. & F. DUGGAN. 1958. A cation car-

rier in the yeast cell wall. Biochem. J. 69: 265-274.

2. EPPLEY, R. W. & C. C. CYRUS. 1960. Cation regu-lation & survival of the red alga, Porphyra perfor-ata, in diluted & concentrated sea water. Biol. Bull.118: 55-65.

3. EPSTEIN, E. 1960. Calcium-lithium competition inabsorption by plant roots. Nature (London) 185:705-706.

4. EPSTEIN, E. & C. E. HAGEN. 1952. A kinetic studyof the absorption of alkali cations by barley roots.Plant Physiol. 27: 457-474.

5. EPSTEIN, E. & J. E. LEGGETT. 1954. The absorptionof alkaline earth cations by barley roots: kinetics& mechanism. Am. J. Botan. 41: 785-791.

6. HANSON, J. B. 1960. Impairment of respiration,ion accumulation, & ion retention in root tissuetreated with ribonuclease & ethylenediamine tetra-acetic acid. Plant Physiol. 35: 372-379.

7. HELDER, R. J. 1958. Studies on the absorption, dis-tribution, & release of labelled rubidium ions inyoung intact barley plants. Acta Botan. Neerl.7: 235-249.

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10. KAHN, J. S. & J. B. HANSON. 1957. The effectof calcium on potassium accumulation in corn &soybean roots. Plant Physiol. 32: 312-316.

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Botan. 8: 36-49.13. TANADA, T. 1955. Effects of ultraviolet radiation

& calcium & their interaction on salt absorption byexcised mung bean roots. Plant Physiol. 30: 221-225.

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OF - · PDF file100 * by flame photometry. The response in terms of K 115 8 absorption paralleled that in the present experiment 215 1 on absorption of Rb. Is calcium the only divalent