Plant Physiol. (1985) 79, 207-2110032-0889/85/79/0207/05/$0 1.00/0

Displacement of Ca2' by Na+ from the Plasmalemma of RootCells'A PRIMARY RESPONSE TO SALT STRESS?

Received for publication February 5, 1985 and in revised form May 9, 1985

GRANT R. CRAMER, ANDRE LAUCHLI*, AND VITO S. POLITODepartments ofLand, Air, and Water Resources (G.R.C., A.L.) and Pomology (V.S.P.), University ofCalifornia, Davis, California 95616

ABSTRACI

A microfluorometric assay using chlorotetracycline (CTC) as a probefor membrane-associated Ca2l in intact cotton (Gossypium hirsutum L.cv Acala SJ-2) root hairs indicated displacement of Ca2 by Na' frommembrane sites with increasing levels of NaCl (0 to 250 millimolar).K+(MRb) efflux increased dramatically at high salinity. An increase inexternal Ca2+ concentration (10 millimolar) mitigated both responses.Other cations and mannitol, which did not affect Ca2+-CTC chelationproperties, were found to have no effect on Ca2`-CTC fluorescence,indicating a Na -specific effect. Reduction of Ca2+_C1C fluorescence byethyleneglycol-bis4W-aminoethyl ether) N,N'-tetraacetic acid, whichdoes not cross membranes, provided an indication that reduction by Na+of Ca2+-CIC fluorescence may be occurring primarily at the plasma-lemma. The findings support prior proposals that Ca2+ protects mem-branes from adverse effects of Na thereby maintaining membrane integ-rity and minimizing leakage of cytosolic K+.

Salt-affected soils make up a substantial portion of the world'sland area including approximately 33% of the irrigated soils (9).Efforts have been made to control salinity by technologicalmeans: reclamation, drainage, use of high leaching fractions, andapplication of soil amendments. An additional approach to theproblem is to utilize the large genetic potential of many cropspecies and select more salt-tolerant crops (3, 9). Selection andbreeding for salt tolerance in a particular crop species would befacilitated if we could identify and understand the mechanismsof salt tolerance and toxicity for that species.Calcium is an important factor in maintenance of membrane

integrity and ion-transport regulation. It has been shown thatCa2" is essential for K+/Na+ selectivity and membrane integrity(8, 14, 18). Elevated Ca2" concentrations in the nutrient solutionmitigated the adverse effects ofNaCl on bean plants by inhibitionof Na+ uptake (12, 17) and reduction in leakiness of membranes(21). LaHaye and Epstein (17) clearly postulated that the Ca2+/Na+ interaction takes place at the plasmalemma. These authorssuggested that Na+ acted by displacing Ca2+ from membranes,leading to increased membrane permeability and intracellularNa+ concentrations.

Additions of Ca2+ salts to a complete nutrient solution partlyalleviated the suppression of root growth in cotton under highsalinity levels (11, 16). Potassium concentrations in the root were

'Supported by National Science Foundation grant DMB84-04442.

reduced by salinity, but were restored to adequate levels by anadditional supply of Ca2" (16). This might indicate that Na+displaced Ca2" from membranes, inducing K+ to leak out of thecytoplasm across the plasmalemma.

Recently, a method was developed to measure chilling-induceddisplacement of Ca2" from membrane sites using CTC2 as aquantitative, microfluorometric probe for membrane-associatedCa2+ (25, 31). CTC, a Ca2+-chelating antibiotic, fluoresces whencomplexed with divalent cations (5). In an apolar environmentthe configuration of the Ca2+-CTC complex changes, as shownby a 5-fold increase in fluorescence emission intensity, while atphysiological pH the fluorescence characteristics of the Mg2+-CTC complex are similar in polar and apolar surroundings (10,13). In addition, the excitation and emission spectra of the Mg2"and Ca2+ complexes are sufficiently different so that with opticalfilters selective for Ca2+-CTC fluorescence changes in the fluo-rescence signal may be considered indicative ofchanges in mem-brane-associated Ca2` (6, 30).

For the study described here, this method was adapted tomeasure changes in membrane-associated Ca2` levels of intactcotton root hairs in response to NaCl treatments at high and low(adequate) external Ca2` concentrations. In this paper, we showthat one of the primary responses to salinity in cells of cottonroots is the displacement of membrane-associated Ca2` by Na+ultimately leading to a disruption of membrane integrity andselectivity as measured by K+(86Rb) leakage from the root cells.

MATERIALS AND METHODS

Plant Material. Cotton (Gossypium hirsutum L. cv Acala SJ-2) seeds were germinated in the dark at 25°C in a VWR 2020controlled-temperature incubator. Seeds were placed in germi-nation paper lining a 1000-ml Pyrex beaker and filled with justenough of a 0.1 modified Hoagland solution (unless otherwisespecified) containing 0.4 mM Ca2" to fully wet the germinationpaper. This Ca2` concentration is adequate for root growth ofcotton under nonsaline conditions (16).

Determination of Membrane-Associated Calcium. Ca2+-CTCfluorescence ofindividual cells was measured using a microscopefluorometer equipped with epifluorescence optics and a voltage-stabilized 100-w DC HBO mercury lamp (Carl Zeiss, Inc., NY).Excitation wavelengths were isolated with a 405-nm bandpass(9.2-nm bandwidth) interference filter; Ca2+-CTC emissions wereisolated with a 530-nm (8.5-nm bandwidth) interference filter(Ditric Optics, Inc., Hudson, MA). Emissions were quantitativelymeasured using a microcomputer-interfaced Hamamatsu R928photomultiplier.

2 Abbreviation: CTC, chlorotetracycline.207

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Plant Physiol. Vol. 79, 1985

Plant experiments were performed using intact cotton roothairs of 8-d-old seedlings. A 2-cm segment from the oldestportion of the root having root hairs was placed in a controlsolution within a custom-built, deep-well microscope slide. Thecontrol treatment solution consisted of0.4 mm Ca2", 50gM CTC,and 2.5 mm T-M (pH 6.5) for all plant experiments unlessotherwise specified. This solution was removed after the fluores-cence determination and replaced with a solution containing thesame reagents as the control, plus the treatment reagent. Cuttingof the tissue had no effect on Ca2+-CTC fluorescence.The deep-well slide was fabricated from 1.3-cm-thick plexi-

glass. Solutions and root segments were held in a groove cut inthe slide and covered with a glass cover slip. No fluorescenceemissions were detected from the plexiglass slide under experi-mental conditions.The microscope fluorometer was focused on individual root

hairs. Background fluorescence was influenced by neighboringroot hair cells. This required measurements to be made at thetips of root hairs to minimize this influence. Care was taken torefocus in the same location of the root hair after the treatmentswere administered. Background fluorescence was subtractedfrom all measurements.

Salt Effect on Ca2-CTC Chelation. A diminished fluorescencecould represent either a decrease in the quantity of membrane-associated Ca2" or a change of the chelating properties of CTC.Since Ca2+-CTC fluorescence in 80% (v/v) methanol has thesame fluorescence maximum (530 nm) as membranes, it wasthought that the properties ofCa2`-CTC chelation in this solutionwere representative of membranes. The effects on fluorescenceof various concentrations of Nae, Ca2", and CTC in 80% meth-anol were measured with a Perkin-Elmer MPF-44A fluorescencespectrophotometer; the excitation and emission wavelengthswere set at 390 ± 10 and 530 ± 5 nm, respectively. The effectson Ca2`-CTC fluorescence of other salts in 80% methanol weremeasured with the microscope fluorometer as described above.

Cellular Location of Ca2-CTC Fluorescence. To assess thecontribution of the cell wall to Ca2+-CTC fluorescence, a rootsegment was treated with 500 mm mannitol. This was sufficientto separate the protoplast from the cell wall in the tips of roothairs, enabling separate measurements of each of these regions.

Treatments with EGTA, a Ca2+-specific chelate, were used toremove Ca2' from the extracellular space, including the externalsurface of the plasmalemma. Ca2+ was left out of the treatmentsolution, but was included in the control solution prior to expo-sure of the root to EGTA.

Cation Effects on Ca2-CTC Fluorescence. Fluorescence ofroot hairs was measured after exposure to treatment solutionscontaining various concentrations of NaCl (O to 250 mM) andCaCI2 (0.4 or 10 mM). The effects of other cations and mannitolwere also examined.KI Efflux. Cotton seeds were germinated in a growth chamber

under the following conditions: 15 h d; photon flux density of200 ME m 2 sl; day:night temperature cycle of 25:20C. Seedswere germinated in a 0.1 modified Hoagland solution lackingthe KNO3 aliquot. Five-d-old seedlings were transferred to thelaboratory (23°C, 15-h d with a photon flux density of 400 MEm-2 s' ) and preloaded with a 0.1 modified Hoagland solutioncontaining 0.6 mm K('Rb)NO3 (pH 6.5) for 3 d.

Roots of intact plants were rinsed for 1 s in 0.4 mM CaS04 toremove residual MRb before placement in the efflux solution.Leakage: of K+(Rb) from intact roots was collected in 15-mlpolystyrene centrifuge tubes containing nonlabeled treatmentsolutions at 23C consisting of various levels of Naa (O to 250mM) in a 0.1 modified Hoagland solution (minus the KNO3aliquot). For the purposes of calculating efflux, the lack ofK+ inthe efflux solution enabled us to assume that K+ uptake wasminimal and therefore the internal speafic activity of K(F86Rb)

remained relatively constant. The amount of K+('Rb) collectedin the centrifuge tubes over various time intervals was determinedby Cerenkov counting in a Packard Tri-Carb 300C liquid scin-tillation system. Semilogarithmic plots oftissue K+(MRb) contentversus time were depicted by a triphasic curve (data not shown).The curve generated was similar in characteristics to those ob-served by other researchers (27). The three phases are interpretedto represent efflux from three cells compartments arranged inseries: cell wall, cytoplasm, and vacuole (27). Samples werecollected over a 0-min interval in the middle of the secondphase. The second phase occurred from 3 to 30 min afterinitiation of tracer efflux and was interpreted to represent effluxfrom the cytoplasm through the plasmalemma.

Chemicals. 86Rb was obtained from New England Nuclear. Allother chemicals were obtained from Sigma.

RESULTS

Salt Effect on Ca2-CTC Chelation. The effect of 150 mmNaCl on Ca2-CTC fluorescence in 80% methanol at various

100

zo so°hi

U~~~~~~~~~~~~ (

a, e

vaiu10ccnetrtos 10 10CC()5 u °,5 ;19 I

(A); mM NaCI (0,0O, A,-), 15S0 mM. NaCI (0,0, t--- -). The size

of the 95% confidence interval bars are no longer than the symbols

except for the 10 MIM CTC treatment as indicated.

Table I. Ca2+-CTC Fluorescence ofan 80% Methanol Solution

Containing 50OMM CTC, 0.4 mMt CaCI2, 2.5 mM Tris-Mes (pH 6.5), and

Various Salts at a Concentration of50 mM

Treatment Ca2--CTC FluorescenceControl (no salt) 54.9 ± 3.9aNaCl 52.5 ± 3.6NaNO3 54.6 ± 2.9LiCI3 58.3 ± 2.7NH4CI 52.7 ± 4.6KCl 56.4 ± 4.2RbCl 55.7 ± 1.8CsC1 58.4 ± 3.8

Control (no salt) 59.5 ± 3.0BaCl2 64.1 ± 5.0SrCl2 74.1 ± 4.3MgCl2 l 16.9 (off scale)MnCl2 36.0 ± 0.6CuC12 25.5± 1.1ZnCl2 r 1 16.9 (off scale)Pb (NO3)2 83.7 ± 11.3LaCI3 ¢1 16.9 (off scale)

I Mean ± 95% confidence interval.

208 CRAMER ET AL.

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DISPLACEMENT OF Ca2+ BY Na+ FROM THE PLASMALEMMA OF ROOT CELLS

Ca2- and CTC concentrations is illustrated in Figure 1. Sodiumdid not affect Ca2-CTC fluorescence except at Ca2+ concentra-tions below 0.4 mm. The Ca2+-CTC fluorescence was also de-pendent upon the Ca24 concentration, but only up to 0.4 mM.The concentration ofCTC influenced fluorescence intensity, butdid not signficantly alter the Na+-Ca2+ interaction.To test the ion-specifity ofCa2` displacement from membranes

by various salts as measured by Ca2+-CTC fluorescence it mustbe verified that these salts do not affect Ca2+-CTC fluorescenceas measured in 80% methanol. The effects of various salts onthe Ca2+-chelation properties of CTC in 80% methanol can beseen in Table I. No monovalent cations had any effect on Ca2+-CTC fluorescence, but di- and trivalent cations at 50 mm affectedfluorescence (except for Ba2+).

Cellular Location of Ca2@-CTC Fluorescence. Fluorescencemeasurements of the protoplast and cell wall of root hairs plas-molyzed in 500 mm mannitol showed that the contribution fromthe cell wall was less than 5% (data not shown). However, thisdoes not represent a quantitative value for Ca2+ in the cell wall.The polarity in the cell wall is different from that of the mem-brane; thus, in the cell wall, CTC has a different affinity for Ca2`and Ca2+-CTC has a different wavelength for maximum fluores-cence. It can be concluded that Ca2`-CTC fluorescence wasprincipally from membrane-associated Ca2+.Treatments with EGTA reduced Ca24-CTC fluorescence of

root hairs (Fig. 2). A large reduction in fluorescence occurredbetween 10-7 and 10' M EGTA. Further reductions in fluores-cence occurred as the EGTA concentration was increased, butto a lesser degree. No cyclosis was observed in root hairs thathad been treated with EGTA above 10-' M.

Cation Effects on Ca2+-CTC Fluorescence. Figure 3 shows thatwith 0.4 mm Ca2` increasing concentrations of NaCl progres-sively reduced the intensity of Ca2+-CTC fluorescence. Attemptsto restore Ca2+-CTC fluorescence by replacing NaCl with 50 mMCaC12 failed to increase fluorescence intensity upon return to acontrol solution. When the roots were exposed to NaCl in thepresence of 10 mM Ca2+, reduction of Ca2`-CTC fluorescenceoccurred only at NaCl concentrations exceeding 25 mm and toa lesser extent than at the lower Ca2l treatment. It should benoted that the data in Figure 3 only represent a relative compar-ison between the low and high calcium treatments. Fluorescencefor the high calcium treatment was greater than the low calciumtreatment. In addition to the results ofthe fluorescence measure-ments, it was observed that with 0.4 mM CaCl2, cyclosis wasinhibited at 150 mm NaCl, but was still visible in solutions

0

-

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z

0-iILUI--U

*1(ti00

I%,_

8

6

4

2

..--71-70 10

-4'o-6EGTA (M)

FIG. 2. Effect of EGTA on Ca2-CTC fluorescence in intact cottonroot hairs. The error bar represents the 95% confidence interval for theentire curve.

2120

o 400 -_

2 1200

0 50 100 IS0 200 2S0 300NoCI (mM)

FIG. 3. Effect of increasing levels ofNaCl on Ca2e-CTC fluorescence(a quantitative measure of membrane-associated Cal2) in intact cottonroot hairs. (O-O), 0.4 mm Ca2"; (0- - .4), 10 mM Ca2l. Each pointrepresents the mean of five replications. The error bars represent 95%confidence intervals for their respective curves.

Table II. Effect of Various Solutes on Ca2'-CTC Fluorescence in IntactCotton Root Hairs

The mannitol concentrations are isosmotic with 50 and 150 mM NaCl,respectively. The external Ca24 concentration was 0.4 mM.

Treatment Ca2tCTC Fluorescence% ofcontrol

5OmMLiCl 101±10a5OmMNaCl 63±650mMKCI 90±950 mM RbCl 102 ± 1950 mM CsCI 103 ± 1650 mM BaC12 107 ± 1396 mM mannitol 101 ± 4269 mm mannitol 96 ± 9

a Mean ± 95% confidence interval.

4-

I.C

-I

IL0

iIL+

4QO

35.0 -

3QLO _25.0 -

20.0 _

15.0-

10.0 ..0

5.0

0.0I I I0 50 100 ISO 200 250 300

NoCI (mM)

FIG. 4. Interaction of Na4 and Ca24 on K+(MRb) efflux rate from thecytoplasm of root cells of intact cotton seedlings. (O-O), 0.4 mm Ca2+;(0- - -4), 10 mm Ca2+. Each data point represents the mean of fourreplications. The error bars represent 95% confidence intervals for theirrespective curves.

209

_ III I I "I I' I I I' I I I

F0

0

1.0

IOU ........... ...... .. ........ ......-- I

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Plant Physiol. Vol. 79, 1985

containing 250 mm NaCI plus 10 mm CaCl2.The effect of those salts which did not affect Ca2+-CTC fluo-

rescence in 80% methanol (Table I) and mannitol on Ca2+-CTCfluorescence in intact root hairs can be seen in Table II. Of allthe treatments, only Na+ significantly reduced Ca2+-CTC fluo-rescence.K Efflux. Leakage of K+(86Rb) from higher plant cells can be

used as an index of membrane integrity. The effect of high Ca2"supply on NaCl-induced membrane leakiness, measured as leak-age from the cytoplasm, is demonstrated in Figure 4. The effluxof K+(86Rb) from the cytoplasm began to increase at NaClconcentrations exceeding 100 mm NaCl. In the low Ca2' treat-ment, effiux rose sharply with further increases in NaCl concen-tration. The response in the high Ca2+ treatment was similar upto 150 mm NaCl, but then diverged dramatically with only aslow rise in K+('Rb) effiux rate.

Ionic effects can be separated from osmotic effects by compar-ison of salt to mannitol treatments. Table III shows that effluxin the mannitol treatment that was isosmotic with 225 mm NaClwas not statistically different from the 225 mM NaCl plus highCa2+ treatment, but significantly lower than the 225 mm NaCItreatment.

DISCUSSION

Under the conditions of this study, Na+ had no effect on theproperties of Ca2+-CTC chelation. Reduction of Ca2+-CTC fluo-rescence by NaCl is interpreted to mean that membrane-associ-ated Ca2" was displaced by Na+. Our results showed that NaClconcentrations as low as 25 mm reduced the quantity of mem-brane-associated Ca2+ when the external Ca2' concentration was0.4 mm. With high external Ca2' supply, root hairs were able tomaintain higher levels of membrane-associated Ca2+ when ex-posed to high concentrations of NaCl. Other cations did notreduce Ca2+-CTC fluorescence, indicating that the reduction byNa+ was ion-specific. It may be that other cations can displacecalcium from membranes at higher concentrations than thosetested in this study, or for those cations which could not be testeddue to their effects on Ca2+-CTC chelation.Of the cations tested, Na+ is the only cation with a crystal

Table III. Effect ofIsosmotic Solutions ofNaCl and Mannitol onK+(86Rb) Effluxfrom the Cytoplasm ofRoots ofIntact Cotton Seedlings

See "Materials and Methods" for details of nutrient solutionsTreatment K+ ('Rb) Efflux

% ofcontrol225 mm NaCl 686 ± 20a225 mM NaCI + 10 mm CaC12 300 ± 133393 mM mannitol 271 ± 149

a Mean ± 95% confidence interval.

Table IV. Physical Chemical Characteristics of Various Cations

Crystal Hydrated Heat ofCation Ion1icRai' Ionic Hydration

Iatninradis Radiib (AH)Ynm kcal/mol

Li+ 0.068 0.37 -121Na+ 0.097 0.30 -95K+ 0.133 0.27 -75Rb+ 0.147 0.24 -69Cs+ 0.167 -61Ba2+ 0.134 -308Ca2+ 0.099 0.44 -377

a Taken from Ref. 7. b Estimated from Ref. 22. c Taken fromRef. 2.

ionic radius similar to Ca2+ (Table IV). The values for thehydrated ionic radii are in considerable dispute (2). Differentmethods ofmeasurement often yield quite different values. Heatsof hydration are reflective ofthe degree of hydration and providea better quantitative measurement (2). The point here is that theresults from the displacement of membrane-associated Ca2" cor-related well with the crystal ionic radii, but not with the param-eters of hydration. This suggests that Na+ may displace Ca2` ina site requiring the ions to be in a dehydrated state. This mayexplain why additions of Ca2` were unable to restore Ca2+-CTCfluorescence after root hairs had been exposed to NaCl. If Ca2+were bound to a specific site, such as in a protein, displacementof Ca2+ by Na+ could alter the conformation of the binding site,since Na+ has a different charge density than Ca2e. This alterationcould then prevent access to the binding site by Ca2 .Hauser et al. (15) suggested that the binding of a given cation

to negatively charged phospholipid surfaces is primarily deter-mined by the charge density and is largely independent of theconformation and hydration of the different polar groups. Theylisted the order of binding for some cations to be:

Ca2+ > Ba2 >> Li' > K+ Na+

Our results do not follow this trend, indicating that the Ca2+-CTC fluorescence monitored in our system reflects binding ofmembrane-associated Ca2+ to ligands other than those in phos-pholipids.EGTA treatments reduced Ca2+-CTC fluorescence (Fig. 2) to

about the same extent as NaCl (Fig. 3). EGTA does not crossmembranes (4, 28), indicating that Na+ displacement of mem-brane-associated Ca2+ may be occurring primarily at the externalsurface ofthe plasmalemma. Interestingly, other researchers (23,31) also were unable to eliminate all the Ca2+-CTC fluorescenceby EGTA in their systems. This residual fluorescence componentmay represent Ca2+ bound to internal membranes, sites on theplasmalemma with greater affinity for binding Ca2+ than EGTA,or background fluorescence ofother di- and trivalent cations thatmay affect CTC fluorescence (Table I).Table III shows that there was an increase in K+(86Rb) efflux

at high salinity and high calcium relative to the control treatment.There was a similar increase caused by an isosmotic concentra-tion of mannitol. In addition, mannitol did not affect Ca2+-CTCfluorescence in root hairs (Table II), so that the increase in effiuxunder these conditions was not the result of Ca2+ displacementfrom the plasmalemma. Since K+(86Rb) effiux was not signifi-cantly different at either the high calcium/salinity or mannitoltreatment, the increase in effiux was probably the result ofosmotically induced changes in plasmalemma permeability. Ta-ble III also shows that K+(86Rb) effiux at high salinity and lowcalcium was significantly greater than at high salinity and highcalcium. This additional leakage of K+(86Rb) can be attributedto ion toxicity to the plasmalemma caused by the high saltconcentration, which can be mitigated by a higher external Ca2+supply.Although it appears that some displacement of membrane-

associated Ca2+ by Na+ did occur in the high Ca2` treatment(Fig. 3), this may not have induced any increase in membraneleakiness. The increase in membrane leakiness in the high Ca2`treatment was most likely due to effects induced by osmoticstress, as discussed in the previous paragraph. It may be that acertain degree ofCa2+ displacement from the plasmalemma mustbe reached before severe changes in permeability occur. Thisconclusion was inferred from experiments in which beet roottissue was exposed to various concentrations ofEDTA (26) andcould explain the differences in salt-stress response observedbetween the two Ca2+ treatments. Alternatively, reduction inCa2`-TC fluorescence by salinity and high calcium may repre-sent displacement of Ca2" by Na+from membrane sites different

210 CRAMER ET AL.

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DISPLACEMENT OF Ca2" BY Na+ FROM THE PLASMALEMMA OF ROOT CELLS

than that at low calcium.The mechanism of the observed K+ leakage is purely specula-

tive. Leakage ofK+ may be the direct result of Ca2" displacementby Na+ from the membrane opening potassium channels. Alter-natively, other indirect factors may be responsible for this re-sponse, such as depolarization of the membrane potential whichcan result from a rise in intracellular Ca2". In support of thelatter hypothesis is the observation that inhibition of cyclosiscorrelated with the large leakage of K+ in the low calciumtreatment, and rises in intracellular Ca2+ concentration havebeen shown to inhibit cyclosis in Chara (29). Na+ displacementof membrane-associated Ca2" could increase membrane perme-ability allowing increases in Ca2+ influx, but at high externalcalcium concentrations this increase in permeability may beminimized.Maintenance of adequate K+ concentrations in root cells is

crucial for continued growth. K+ is needed not only for cellturgor to drive cell expansion, but also as a cofactor for manyenzymes (20). Furthermore, the leakage of K+ from the celllowers the K+/Na+ ratio in the tissue (16). K+/Na+ selectivityhas been found to be an important factor in the salt tolerance ofcotton (19). The data presented in this study strongly indicatethat high Na+ concentrations displace Ca2" from the plasma-lemma, resulting in a loss of membrane integrity and efflux ofcytosolic K+.Young and Kauss (32) attempted to study the effect of poly-

cations, polyamines, and polyanions on cell surface calcium, andtheir relation to membrane permeability using 45Ca2+ to measureCa2" displacement. They found that the amount of 45Ca2' dis-placed from isolated cell wall fragments was similar to wholecells and that permeability did not correlate with 45Ca2' displace-ment. These effects are difficult to interpret, because it is notclear whether cell wall and/or plasmalemma Ca2` was displaced.Since the cell wall contains the largest proportion of total cellcalcium (14), we suggest that a large displacement of Ca2' fromthe cell wall would mask any displacement from the plasma-lemma. Their data showing displacement of 45Ca2' by differentcations cannot be compared to ours for the reasons stated aboveand because they used much lower cation concentrations (0.5mM). Clearly, our method was capable of distinguishing thesetwo components. However, the Ca2+-CTC fluorescence tech-nique appeared to be unsuitable for studying the effects of mostdi- and trivalent cations on membrane-associated Ca2+, sincethese cations also affected the properties of Ca2+-CTC chelation,at least at the concentrations used in this study.We suggest that the displacement of membrane-associated

Ca2' by Na+ is a primary response to salinity in this cottoncultivar. Whether or not this holds true for other cultivars orspecies remains to be verified. Recently, differential growth re-sponses to salinity as affected by external calcium have beenobserved within a species (1, 24). It would be interesting to knowif these differences can be attributed to differences in displace-ment of membrane-associated Ca2` or to some other factor.

In summary, Na+ displaced membrane-associated Ca2+ fromcotton root hairs. This response was Na+-specific and may beoccurring primarily at the external surface of the plasmalemma.Both the displacement of membrane-associated calcium and theincrease in membrane leakiness were mitigated by a high externalCa2" supply.

Acknowledgments-We thank Dr. E. Epstein and J. Lynch for critical readingof this manuscript, and the latter for his suggestions and assistance in the effluxstudies.

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32. YOUNG DH, H KAUss 1983 Release of calcium from suspension-culturedGlycine max cells by chitosan, other polycations, and polyamines in relationto effects on membrane permeability. Plant Physiol 73: 698-702

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