Plant Physiol. (1980) 65, 544-5490032-0889/80/65/0544/06/$00.50/0

Effect of Salinity upon Cell Membrane Potential in the MarineHalophyte, Salicornia bigelovii Torr.

Received for publication March 20, 1979 and in revised form November 1, 1979

ALAN L'RoY AND DONALD L. HENDRIXDepartment of Biology, University of Houston, Houston, Texas 77004

ABSTRACT

The electrophysiology of root cels of the marine halophyte, Salicorniabigelovii Torr., has been investigated. CeDlular concentrations of K+, Cl1,and Na+ and resulting cell membrane potentials were determined asfunctions of time and exposure to dilutions of artificial seawater. Treatmentof these data by the Nernst criterion suggests that Cl is actively trans-ported into these root cels, but that active transport need not be invokedto explain the accumulation of Na+ at all salinities investigated nor for K+at moderate to high sallnities. In low environmental salinity, the cellelectropotential of Sal;cornia root cells was found to respond to inhibitors -

in a fashion similar to that observed in glycophytes; in high environmentalsalinity, root cel membrane potential appears to be insensitive to bathingsalinity and m-chlorocarbonylcyanide phenylhydrazone induces membranehyperpolarization, in contrast to the response of glycophytes to suchtreatments. The fact that measured membrane potentials exceed diffusionpotentials for Na+, K+, and Cl and the observation of a rapid depolari-zation by CO in the dark suggests an electrogenic component in Salicorniaroot cell membrane potentials.

The genus Salicornia (Chenopodiaceae) includes a number ofsucculent euhalophytes (24) which show differences in salt toler-ance; some members of this genera are among the most saltresistant of higher plants (5, 24, 25). We have found that theannual Salicornia bigelovii will grow in a wide range of salinitiesin laboratory culture (22) and that results with laboratory-grownmaterial agree very well with those obtained from plants collectedin the field.Although halophytic marine higher plants have been investi-

gated for many years, our knowledge of their environmentaladaptations is limited. Their unique ability to transport mineralions in the presence ofhigh concentrations ofsodium and chlorideis critical to their survival and yet poorly understood; furthermore,of the major driving forces for inorganic ions, the PD' has beenlargely unexplored in these species. Most of the literature in thisarea concems the PD of salt glands (4, 11, 19); far less attentionhas been devoted to the roots of halophytic plants as absorbingorgans with unique adaptations to their severe environment (see3, 16).

In the present paper we report the effect of environmentalsalinity upon cell K+, Cl-, and Na+ content and cell membranePD in S. bigelovii root cells. Data so obtained have been analyzedby the Nernst criteria to provide an estimate of the energetics ofthe absorption of these monovalent ions by Salicornia root cells.

' Abbreviations: PD: cell transmembrane electropotential; ASW: artifi-cial seawater (Instant Ocean, Aquarium Systems, Inc., Eastlake, Ohio);CCCP: m-chlorocarbonylcyanide phenylhydrazone; EN: calculated Nernstpotential; EM: measured cell electropotential.

MATERIALS AND METHODS

Plant Material. Plants containing mature seeds were collectedfrom homogeneous stands along the beach and surrounding marshland near Galveston, Texas. Seeds were removed from these field-dried plants by scraping and were stored at 4 C until needed.Seeds were germinated upon cheesecloth placed in illuminatedshallow enamel trays for 7 days (or more, in some instances) at 23C; the cheesecloth was watered with distilled H20 to start germi-nation and the tray covered with transparent plastic to retardevaporation. At the end of this period, seedlings were transferredto Vermiculite and thenceforth watered with a given concentrationof ASW. ASW was prepared by dissolving 1.47 kg of InstantOcean salts mixture (Aquarium Systems, Inc.) in distilled H20 togive a final volume of 37.85 liters; 15.9 ml of liquid trace elementswere added after dissolution of the salt (23). The major compo-nents of this solution (designated 100%o ASW), in mm, are: 519C-, 444 Na+, 6.0 4o42-, 49.4 Mg2+, 9.5 K+, 9.2 Ca2+, 2.3 HC03-,0.4 H3BO3, 0.25 Br-, and 0.09 Sr2+. For a complete listing of thecomponents of this solution, see reference 23. Dilutions of ASWwere prepared by diluting 100%o ASW with distilled H20. The pHof 100%o ASW and its dilutions was found to be approximately7.6.

Seedlings were grown under light banks consisting of a mixtureof General Electric F48PG 17 CW, Sylvania F48T 12-VHO GROWS, and Duro-Test 48T12 HO 800 mamp Vita-Lite fluorescentbulbs (10.5 ,uw cm-2 nm-' at 655 nm) timed to give a 14-hdaylight/10-h night regime. A diminished light, created by dou-bling the distance between the seedlings and the bulbs, was utilizedduring the first 7 days of plant growth to retard evaporation.

Electrical Measurements. Root tips approximately 1.0 cm longwere removed from plants 7-30 days old with a razor blade andinserted into a micro flow chamber placed upon a microscopestage (6, 20). Before impalement, excised root tips were held inaerated ASW of the same composition as that used for electricalmeasurement. The microchamber was perfused with varying di-lutions of ASW by gravity flow. Drastic changes in ASW concen-tration during electrode impalement led to poor electrical meas-urements, possibly due to the alterations induced by such changesin cell turgor pressure; therefore, tissue was allowed to equilibratefor 10-15 min to any change in perfusate before electrical meas-urements were recorded. Contrary to observations on glycophytecell PD (20, 21), the measured PD in Salicornia root cells did notappear to change with time during the first 5-6 h followingexcision. Tissue was discarded if, under microscopic examination,it appeared damaged or had been excised for more than 3 h.

Cell potentials were recorded using Ling-Gerard glass capillarymicro salt bridges and silver/silver chloride electrodes, as detailedelsewhere (20, 21). Glass salt bridges were pulled from capillarytubing containing glass fibers (Omega Dot capillary tubing, Fred-eric Haer Co., Ann Arbor). Electrodes used had resistances of lessthan 10 megohm in 100% ASW, and were changed frequentlyduring recording sessions. Cells used for electrical measurementswere chosen from cortical cells in the same region of the root tip,

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ELECTROPHYSIOLOGY OF A MARINE HALOPHYTE

approximately 100-200 im above the root cap, since PD valueshave been found to vary between different regions of plant roots(20). The potential difference between an agar-filled referenceelectrode placed in the bathing medium and an electrode placedwithin a cell by micromanipulation was amplified by a WinstonElectronics model 1090 microelectrode preamplifier and recordedwith a strip chart recorder.CCCP and CO Solutions. CCCP solutions were prepared by

dissolving CCCP in absolute ethanol and diluting this stockimmediately before use (18) to 1 x 10-5 M with ASW by directinjection of a small amount of the ethanol stock into the perfusionchamber. For control purposes, ASW containing an equivalentamount of ethanol was perfused through the chamber; ethanolitself, at this dilution (1%, v/v), had no effect on cell PD. COsolutions were prepared by saturation ofASW dilutions with C.P.grade CO by vigorous bubbling (10). These CO-saturated ASWsolutions were injected into the microchamber after stopping theinflux of nonpoisoned ASW.

Tissue Ion Content. Seedlings were separated into roots andshoots by cutting with a razor blade, the tissue thoroughly rinsedin distilled H20 to remove adhering Vermiculite and salt, weighedafter blotting with filter paper, and extracted with boiling water.K+ and Na+ in the water extracts were then determined by flameemission spectroscopy and Cl- determined by amperometric titra-tion.

RESULTS

The PD of Salicornia root cells changes over a 2 week periodfollowing the transfer of 1-week old seedlings from distilled H20to varying dilutions of ASW (Fig. 1). The root cell potential ofseedlings exposed to 10%1o ASW falls strongly and the internalionic concentration increases with time (Table I); however, thisdrop in membrane potential does not appear to be a simplefunction of increasing internal salinity since the PD of root cellsexposed to 100%1o ASW exhibit a slight but significant increase(F1,3 = 11.9, P < 0.05) even though these cells' ionic content alsoincreases to levels higher than that in seedling roots at the start ofthe ASW exposure period (Table I). The intermediate salinitiestested exhibit a gradual transition between these two responses.The change in root cell membrane potential at all salinities appearsto be most pronounced during the first 2 weeks following transferfrom distilled H20 (Fig. 1). We investigated this period more

closely as a possible time of adjustment of the plants to the stressof high environmental salinity.A determination of the ionic content of plant roots 7 days after

transfer to Vermiculite and the start of the ASW exposure indi-cated that the Na+ and K+ content of these seedlings is relativelyconstant despite the wide range ofenvironmental salinity to whichthey were exposed (Table I). The Cl- content of these roots showsan apparent increase with environmental salinity, the correlationbetween environmental and internal chloride having an r value of0.916.

-180

-14 0

-. -1 00'

- -1800

a.

-140

Lu

-1 0

-180

-14 0

-100

10% 50%

2 On*. e o0 *

_~~~~~~~~~~~~~~~~~~~~~~~~~100%3 0 ,0

2 1 0 20 2 10 20Day s

FIG. 1. Change in the PD of Salicornia root cells with time ofexposureto various dilutions ofASW; percentages refer to dilution of Instant Ocean.Seedlings were germinated on cheesecloth watered with distilled H20 forI week prior to time of transfer to ASW dilutions and Vermiculite culture;day zero of figure represents day of transfer to Vermiculite watered withappropriate ASW solution. Bars represent :SD, points represent means ofthree to nine replicates.

Table I. Ionic Content of Salicornia Seedlings, 14 Daysfrom GerminationSeedlings transferred to ASW dilution indicated 7 days after germination. Values represent means of three replicates ± SD. Superscript 0 denotes

concentration in bathing medium of ion indicated in mm.

ASW Root/ Na° Na+ EN K° K+ EN ClO Cl_ EN EM RootShoot

% mM ,umol/gfresh wt mv miM pimol/gfresh wt mv mM pmol/gfresh wt mv0 R 96

S 9410 R 44.4 164 ± 2.5 -33.3 0.95 147 ± 3.6 -129 51.9 170 ± 3.6 30.3 -159

S 96±3.1 43±4.0 184±4.720 R 88.8 220 ± 4.0 -23.4 1.90 130 ± 4.0 -108 104 -158

S 140± 3.5 54±3.2 240± 8.530 R 133 230 ± 4.7 -14.0 2.85 150 ± 4.2 -101 156 250 ± 5.0 12.0 -153

S 173±4.0 58±2.5 281±5.050 R 222 240 ± 3.0 -0.2 4.75 142 ± 1.4 -86 260 269 -0.9 -135

S 217 60±3.1 30880 R 355 183 ± 4.5 16.9 7.60 135 ± 3.6 -73 415 285 ± 4.7 -9.6 -133

S 298±2.0 56±3.0 392±5.7100 R 444 270 ± 4.1 12.7 9.50 115 ± 0.7 -64 519 310 ± 2.8 -13.1 -133

S 364 ±4.5 64 ± 3.6 480

t b.

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Plant Physiol. Vol. 65, 1980

The K+ content of the shoot is relatively constant over the rangeof ASW dilutions tested and noticeably lower than that of theroots; shoot Na+ and Cl- content, however, increases strongly withincreasing salinity (Table I). In fact, there is a strong positivecorrelation between both the Na+ content of the shoot and envi-ronmental Na+ (r = 0.966) and the Cl- in these cells versus externalCl- (r = 0.988). High shoot Na+ appears to be characteristic ofhalophytes in general (9).We did not find drastic changes in root salt content to occur

between 15 and 24 days from germination (Tables I and II), aperiod which corresponded to significant PD changes in rootcells. A slight increase in root cell content during these 10 days isaccompanied by a drop in CF- content and an accompanyingslight drop in Na+ in most ASW concentrations tested; the con-verse being true for the cellular concentrations of these ions in theshoot (Tables I and II). Since this pattern of cell ion contentchange is roughly the same across all salinity values investigated,it does not seem that root cell electropotential changes can beexplained by changes in internal salt concentration alone. Alsonote that during this time period, growth of both the shoot androots of this plant was very slight.To further explore Salicornia root cell PD behavior, we em-

ployed metabolic inhibitors to estimate the active contributions tocell PD and the Nernst criterion to arrive at the diffusion potentialsofthese three monovalent ions. The Nernst equation is representedby:

RT [K+]0 RT [Na+]0 RT [CF-]0EN = - [K+]i = zF z[Na+]zF [Cl-],

where EN = Nernst potential in volts; R = universal gas constant;T = absolute temperature; F = Faraday; z = ionic valence, andsuperscripts, i and o, refer to activities (concentrations) of ionsinside and outside the cell, respectively. This relationship can beused to estimate a diffusion potential between the cell interior andthe bathing medium for each of the ions considered under con-ditions of independent, passive ion movement and flux equilib-rium between root cells and the root environment (13, 14). Pre-dicted PD values for each ion can then be compared to measuredelectrical potentials (Tables I and II). Such comparisons in non-photosynthetic glycophyte cells usually show the PD of such cellsto be close to a diffusion potential for K+; i.e. for most nonpho-tosynthetic glycophyte systems, a 10-fold increase in the ratio ofK+ outside the cell to that inside causes a -58 mv change in PD(12, 21). This is clearly not the case in Salicornia root cells for anyof the three ions tested (Tables I, II, and IV).

Using a converse approach, one may compare the actual ioncontent of the tissue with that predicted by the Nernst relation. Inboth 14- and 24-day old seedling roots exposed to ASW for 7-17

days, respectively, predicted values for K+ are usually higher thanthose measured, values for Na+ always much higher and valuesfor CF- are also far higher than those predicted by the Nernstequation (Table III). In low ASW concentrations, K+ approachesits predicted concentration, but the strong increase in cell K+content predicted by the Nernst relation for increasing ASWconcentrations does not occur. These data suggest that CF- isaccumulated by Salicornia roots at the expense of metabolicenergy in all dilutions of ASW investigated; however, metabolicenergy need not be invoked to explain the accumulation of Na+or for K+, except for the lowest ASW concentrations investigated(Table III).

Since the PD in these roots does not seem, in general, to respondin a fashion suggesting a relation to the passive distribution ofNa+, K+, or CF-, and because the root PD responds so differentlywith time at high and low ASW dilutions, we decided to investigatethe active components of cell PD from plants grown in 10 and100o ASW by means of metabolic inhibitors. Cell PD, measuredin either young (Fig. 2, A and B) or older (Fig. 2, C and D)seedling roots, responds in a similar fashion to the uncouplingagent CCCP; at low external salt concentrations, a depolarizationof root cell PD results, but at high salt concentrations, a hyper-polarization results. A much different result has been found forthe respiration blocking agent, CO (Fig. 3); blocking electrontransport by infusing the tissue with CO and forming the lightreversible iron carbonyl complex (and supposedly stopping respi-ration) by turning off the microscope illuminator (2, 10), causes adepolarization of cell PD in either high or low ASW concentra-tions.The response of Salicornia root cell PD over a short time period

to changes in external salinity is quite different from that foundfor glycophytes. Changing the salinity of the solution bathingroots taken from plants grown in 100%o ASW causes a rise inmeasured electropotential values, when the bathing salinity in-creases from 20 to approximately 50%o ASW; at higher salinity,measured values remain relatively constant (Table IV).

DISCUSSION

In low salinity, the PD of Salicornia bigelovii root cells appearsto behave in a manner similar to that of glycophyte cells (1, 2, 8,10, 13, 20), and markedly different from the behavior of glyco-phyte cells in increasing salinity. The membrane PD of pea stemcells, for example, is depolarized both by 10 !lM CCCP andsaturated aqueous CO in the dark (2, 10) in much the same waythat the PD of Salicornia roots behaves in 10%1o ASW. The hyper-polarization of Salicornia cell PD by 10 Atlm CCCP in high externalsalinity, however, appears to be a departure from glycophytebehavior (Fig. 2). It is not known whether this hyperpolarization

Table II. Ionic Content of Salicornia Seedlings, 24 Daysfrom GerminationSeedlings transferred to the ASW dilution indicated 7 days after germination. Symbols as for Table I.

ASW Root/ Na° Na' EN KO K+ EN ClO Cl- EN EM RootShoot% ~ ~ ~ fmm ,smol/g fresh wt my mm. pmol/gfresh wt "ii 'mm u.mol/gfresh wt my

44.4 101+2.186

88.8 190± 2.1139 + 3.5

133 180 ± 9.1139 ± 4.2

222 189 ± 4.2189 ± 4.2

355297 ± 14.1

444 140 ± 9.8154 ± 7.0

-20.1 0.95 157 + 8.152 + 6.4

-19.4 1.90 92 +4.932 4.2

-7.7 2.85 160 ± 5.0

35 ± 5.74.1 4.75 163 ± 3.5

40± 5.0

7.6036 ± 5.7

29.4 9.50 167 ± 5.457 ± 5.5

-130 51.9 140 ± 4.9160 ± 7.8

-99 104 191 ± 6.6192 ± 3.5

-103 156 199± 11.8229 ± 4.2

-90.2 260 248 ± 6.6246 ± 7.1

415 273 ± 9.2364 ± 5.7

-73.2 519 265 ± 4.2350 ± 2.1

25.3 -118

14.1 -140

6.2 -145

-1.2 -142

-10.7 -139

-17.1 -149

10 RS

20 RS

30 RS

50 RS

80 RS

100 RS

546 L'ROY AND HENDRIX

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ELECTROPHYSIOLOGY OF A MARINE HALOPHYTE

Table III. Prediction of Ionic Contents of Salicornia Roots by NernstRelation

Superscript' refers to internal ion content, other symbols as for Table I.

ASW Ion EM [ionl [ionl]'meas. [ionJpred.% mv mm

14-Day-Old Roots10 K+

Na+Cl-

20 K+Na+Cl-

30 K+Na+Cl-

50 K+Na+C1-

80 K+Na+Cl-

100 K+Na+Cl-

10 K+Na+C1-

20 K+Na+C1-

30 K+Na+C1-

50 K+Na+C1-

80 K+Na+C1-

100 K+Na+C1-

-159 0.9544.451.9

-158 1.9088.8104

-153 2.85133156

-135 4.75222260

-133 7.60355415

-133 9.50444519

24-Day-Old Roots-118 0.95

44.4051.90

-140 1.9088.8104

-145 2.85133156

-142 4.75222260

-139 7.60355415

-149 9.50444519

147164170130220

150230250142240269135183285115183310

157101140152190181160180199163189248

273167140265

48522,664

0.102933

43,5840.212

1,15053,656

0.387946

44,2141.306

1,39965,369

2.2501,825

81,7572.82

97.14,540

0.533460

21,5160.429

84039,208

0.5291,245

58,1800.992

1,77182,710

1.8693,276

161,3941.505

is a phenomenon peculiar to Salicornia, to halophytes, or to highexternal salinity, but it is apparently not related to the age of thetissue (Fig. 2).The hyperpolarization by CCCP but depolarization by CO at

high salinity (Figs. 2 and 3) suggests a different mode of actionfor these two inhibitors. The common assumption is that plantcell membrane potentials are a composite of a diffusion potential,due to the passive distribution of ions, and an active electrogenicpotential due to the metabolic pumping of ions across the cells'limiting membranes (13, 15). The electrogenic pump is usuallythought to be driven by ATP breakdown (13, 15) and conditionswhich decrease the ATP pool in cells should eliminate this voltagecomponent. Both CCCP and CO (in the dark) have been suggestedto lower cytoplasmic ATP levels in other organisms. If they do soand the electrogenic pump depends upon ATP, then one shouldexpect a similar effect on membrane potential, and, in fact, theydo have a similar effect upon cell PD in glycophyte tissues (2, 11).However, CCCP is also known to increase in membrane permea-bility, especially with respect to H+ (18) and Cl- (17). Therefore,the hyperpolarization caused by CCCP at high salinity valuescousd involve an increase in cell membrane permeability. For

200r

E

0-

(13

G)

wL

U)

0 1 0 20 30 40 50M;i n U t e s

FIG. 2. Effect of 10 pM CCCP on the membrane PD of Salicornia rootcells of varying ages and salinity during growth. A and B represent datafrom seedlings exposed to ASW for 7 days; C and D represent data fromseedlings exposed to ASW for 21 days. Ten pm CCCP were added to ASWdissolved in a small amount of alcohol (1%, v/v); Con refers to theintroduction of an equivalent amount of ethanol. ASW refers to the returnof perfusion of nonpoisoned ASW. Representative tracings of severalreplications are shown.

Table IV. Responses of Salicornia Root Cell Electropotential to Changesin Bathing Salinity

All cells taken from roots 20-21 days from germination, 13-14 daysafter transferring to Vermiculite watered with 100%1o ASW. Cells wereallowed to equilibrate in diluted ASW before excision for approximately15 min before impalement. Data represent means ± SD of three to ninereplicates.

ASW Bathing Root EM% mv20 -115 730 -130 750 -152 4.280 -147 5100 -149 6

example, if CCCP increased the membrane permeability to H',the resulting PD changes observed in Figure 2 could be explainedby an internal pH of approximately 6.5 in 10%o ASW and 4.5 in100%o ASW. In fact, acidification of plant cell cytoplasm in re-sponse to salinity has been observed (9).Another possibility might entail a low concentration of Cl- in

the cytoplasm that would present a low EN for this ion in lowsalinity and a high EN in response to high salinity. If CCCP wereto increase the plasma membrane permeability to CF-, this could

Plant Physiol. Vol. 65, 1980 547

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L'ROY AND HENDRIX

100

off

t ~ton on

100/0 ASW

L

1 00

ofASW

onon

1000/o ASW

0 10 20 30M i n U t e S

FIG. 3. Effect of ASW saturated with CO upon Salicornia root cellmembrane potential. The microscope illuminator was switched off or onat the points indicated in the figure. CO represents the point ofintroductionof ASW saturated with CO; ASW represents the point of introduction ofnonpoisoned ASW. Typical tracings of several replications are shown.

lead to a membrane hyperpolarization if external Cl- was highand a depolarization of external Cl- was low.

In both of these models, the sustained nature of the hyperpo-larization is difficult to account for; ie. why does the cell remainhyperpolarized for up to 20 min (Fig. 2) when (supposedly) onlydiffusion is operating? Why is the hyperpolarization not transient,as is sometimes experienced with CN- inhibition (2)? Under COpoisoning (Fig. 3) the potential reached most closely approachesthat of a K+ diffusion potential, of those considered (Fig. 3 andTable I), as if, under CO poisoning, EM depends largely upon K+.The fact that Cl- and Na+ do not appear to be near passive

equilibrium (i.e. that predicted by the Nernst equation) betweenthese cells and the bathing medium should not be surprising, sincethese ions are usually found to be removed from equilibrium inglycophytes as well (14). On the other hand, the glycophyteliterature suggests that K+ is usually closer to equilibrium valuesthan we have found for Salicornia roots. These discrepanciesbetween actual and equilibrium values could be due to a numberof factors, including active pumping of the ions in question andcompartmentation of salt within the tissue; thus, the ionic contentof the component in which the electrical measurements wereperformed may differ from those estimated here. The tip of theelectrode, for instance, might be in the vacuole whose ion contentand, therefore, the PD measured, might differ from measurementsresulting from an electrode tip placed within the cytoplasm. Inglycophytes, at least, this possibility does not seem to be a problem,since the potential difference across the tonoplast is usually foundto be slight and vacuolar PD measurements thus closely reflectcytoplasmic values (7 and refs. therein). It is not possible to

determine the contribution of cytoplasm/vacuolar ion contentdifferences from the present data.Some ion besides the three investigated could also be involved

in determining the membrane PD in Salicornia root cells. Theuncoupler data discussed above would suggest H+ as a distinctpossibility.Root cells of the halophyte S. bigelovii exhibit cell PD values

which differ in several respects from equivalent measurements onglycophytic species. Some of the deviation from glycophytic be-havior, such as hyperpolarization by CCCP, appears to be foundin the presence of high external salinity. Another deviation is thelack of decrease of cell PD with increasing environmental salinity.Whether these differences from glycophyte behavior are relatedto the adaptation of this plant to its environment must awaitfurther experimentation.

Finally, CF- is apparently accumulated at the expense of meta-bolic energy by Salicornia roots in all environmental salinities;Na+ accumulation at all salinities and K+ accumulation at all butthe lowest salinities investigated does not appear to require met-abolic energy. The fact that EM exceeds EN for Na+, K+, and CF-and the rapid depolarization induced by CO in the dark suggeststhat Salicornia root cell PD contains an electrogenic componentas has been suggested for glycophyte cells (1, 12, 13).

Acknowledgments-We wish to thank Drs. N. Higinbotham, C. B. Osmond, B.Etherton, U. Luttge, R. M. Spanswick, and W. S. Pierce for helpful suggestionsduring the preparation of this paper.

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E

0~

(U

L-

U-uJ

UL

548 Plant Physiol. Vol. 65, 1980

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Plant Physiol. Vol. 65, 1980 ELECTROPHYSIOLOGY OF A MARINE HALOPHYTE 549

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