Plant Physiol. (1982) 70, 709-7130032-0889/82/70/0709/05/$00.50/0

UV-Stimulated K+ Efflux from Rose CellsCOUNTERION AND INHIBITOR STUDIES

Received for publication March 9, 1982 and in revised form May 15, 1982

TERENCE M. MURPHY AND CLYDE WILSONDepartment of Botany, University of California, Davis, California 95616

ABSTRACT

Irradiation of a washed suspension of cultured rose (Rosa damascenavar. Gloire de Guilan) cells with about 1,680 joules per square meter ofshort wave ultraviolet (UV) light (254 nanometers) caused K+ to appear inthe external medium. Short-term tracer (86Rb+) experiments confirmedthe earlier suggestion (Wright, Murphy 1978 Plant Physiol 61: 434-436)that UV increases the efflux of K+; there was also a small decrease ininflux of K+. There was a partial recovery of fluxes from the effects of UVradiation, but no net accumulation of K+ within 16 to 18 hours after theirradiation. The K+ appearing in the medium was matched by an equivalentamount of HC03-; it was suggested that HC03- was the principal coun-terion for the K+ flux induced by UV. Inhibitors of ATP synthesis (10-5molar carbonyl cyanide m-chlorophenyl hydrazone; 0.05 millimolar KCNplus 0.75 millimolar salicylhydroxamic acid) strongly reduced the UV-stimulated K+ leakage, suggesting that the leakage was dependent in someway on ATP concentration inside the cells. The UV-induced K+ leakagewas also dependent on temperature and the presence of Ca2+ in theexternal medium.

A number of reports have shown that short wave UV radiation(<280 nm) causes an increase in the permeabilities of plant cells.The irradiation of beet roots with a high fluence (20,000 J m-2)causes sufficient disruption to allow betacyanin to leak out of thecells (15). High fluences also cause changes in the ultrastructureof pea and tobacco chloroplasts (1, 16). Fluences >1800 J m-2 leadto peroxidation of lipids from wheat roots measured by malondi-aldehyde production (24) which may account for the changes inpermeability and ultrastructure. Lower fluences of UV inducemore specific changes in the permeabilities of cells to ions.Doughty and Hope (3), working with Chara corallina, found thatUV (< 1400 J m- ) depolarized the membrane (plasmalemma plustonoplast) and decreased its resistance, while it stimulated atransient efflux of Cl-, K+, and Na+. Wright and Murphy (23)reported that fluences of UV <1600 J m-2 caused washed, prela-beled tobacco cells to lose '6Rb+, though they did not stimulateinflux of [3H]mannitol or efflux of 2-[3HJdeoxyglucose. The spe-cific changes in ion permeability are particularly interesting, be-cause they may explain in part the pathological effects of UV onplant tissues (Wright and Murphy, in press), and conceivably theycould be related to nonpathological effects of UV on the differ-entiation of plant cells (4).

There is as yet little information on how low fluences of UVinfluence higher plant membranes. Wright et al. (24) reported thatUV inhibits the activity of the plasma membrane K+-stimulatedATPase, an enzyme thought to be involved in K+ accumulation(I1). But the original work of Wright and Murphy (23) did notspecify whether UV affected influx or efflux of the 'Rb+. It also

did not identify the counterion accompanying the net movementof the monovalent cation, or determine whether the flow wasdependent on active energy metabolism. The work reported inthis paper fills some of these gaps.

MATERIALS AND METHODS

Cells. Cell aggregates of Rosa damascena var. Gloire de Guilanwere subcultured every 7 d by a 1:10 dilution in MXG medium(see Ref. 13 for details). Experiments reported below used 5-d-oldcultures, which had approximately 3 x 106 cells/ml distributed inaggregates. The suspension was fine: few or no aggregates werelarger than 0.5 mm in diameter. At this stage, packed cell volumewas about 0.2 ml/ml of suspension, but the wet weight anddisplacement volumes of filtered cells indicated that the trueprotoplast volume was less than 0.06 ml/ml of suspension. Beforeeach experiment, cells were filtered three times and resuspendedin a volume of 0.01 M CaCl2 equal to the original volume ofsuspension. After the final wash, 10-ml volumes (3 x 107 cells in10 mm CaCl2) were distributed into 100-ml beakers.

Ion Concentrations. The concentration of K+ in the cell suspen-sion was measured by placing a K+-sensitive glass electrode(Markson) and a separate calomel reference electrode directly inthe suspension. The suspension was gently swirled and thenallowed to settle. A stable reading was observed about 10 to 20 safter the swirling. The electrode combination and meter werecalibrated each day with 10-4, 10-3, and 10-2 M KCI. The concen-tration of Cl- in the cell suspension was measured in a similarfashion using a solid-state Cl--sensitive electrode (Orion). Ca2+,NO3 , P043 , orffanic acids, and HCO3 were measured in cell-free filtrates. Ca + was measured by atomic absorption spectrom-etry (Perkin-Elmer). N03- was measured by HPLC (18). P043-was measured by the method of Fiske-SubbaRow (6). Organicacids were separated by HPLC (Bio-Rad HPX-87 ion exclusioncolumn with 35 mm H2SO4 liquid phase) and detected by OD at210 nm; malic, citric, succinic, and oxalic acids were used asstandards. Titrations of acids and their conjugate bases wereperformed in filtrates that had been quickly overlaid with mineraloil to prevent exchange of CO2 with the atmosphere. Estimates ofHCO3 amounts were calculated from titration curves by assumingthat 94% of the anion became associated with H+ between pH 7.9and pH 4.9.

Influx and Efflux. Influx of 'Rb+ was measured by adding 0.5,uCi of [86Rb]Cl (Amersham-Searle, >86 mCi/mmol) to 10 ml ofwashed cells immediately after measuring K+ of the suspension.One-ml samples taken 1, 3, 6, and 10 min later were each filtered,washed with 30 ml of solution containing 20 mm KCI and 10 mMCaCl2, and dried. The washing took approximately 1 min. Driedfilters were counted in 4 ml of dioxane-based scintillation cocktail(125 g naphthalene, 7.5 g 2,5-diphenyloxazole, 0.375 g of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene/I of dioxane) in a liquidscintillation spectrometer (Beckman model LS-8000). To estimatetotal activity, 0.2 ml of suspension were absorbed onto a filter disk

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MURPHY AND WILSON

without washing. Influx was calculated by the slope of the regres-sion line relating the radioactivity of the four samples to the timeof sampling, divided by the total activity concentration and mul-tiplied by the measured K+ concentration.

Efflux of 86Rb+ was measured by adding 50 ,uCi of ["6RbJCl to50 ml of cell suspension 12 h or more before washing the cells.Cells were washed as usual, distributed in l0-ml aliquots, andallowed to incubate. At the desired time, they were rewashed intomedium containing a known concentration of KHCO3. One-mlsamples taken 1, 5, 10, and 15 min later were filtered. The cellswere discarded and 0.2 ml of the filtrate was mixed with 4 ml ofdioxane-based scintillation fluid for counting. The initial radio-activity and K+ concentration in the cells were estimated byfreezing and thawing the cells three times and sampling theresulting suspension. Calculations were performed as describedabove.

Respiration. The production of CO2 by cell suspensions wasmeasured by IR gas analysis. Ten ml of suspension were placed ina closed cylindrical chamber (4.0 cm diameter x 2.7 cm deep).The top of the chamber was either a piece of UV-transparentplastic (for irradiations) or a piece of plastic with a syringe port(for inhibition experiments). The suspension was agitated byplacing the chamber on a shaker. Air treated with Drierite andAscariteII was introduced into the chamber at a measured rate(0.35-0.38 1/min). Effluent air was passed over Drierite andthrough the sample chamber of the analyzer (Beckman model865). The same air introduced into the chamber was also directedto the reference chamber of the analyzer. The analyzer was

calibrated by comparing reference air to air that was not AscariteII-treated (assumed 320,ul/l C02). The response time for detectingchanges in respiration was rapid (within 60 s) as judged by thefastest responses to the addition of uncoupling agents.02 uptake was measured using a Rank Brothers, Clark-type

electrode (5). After appropriate washing and chemical or radiationtreatments, 5 ml of cell suspension were placed into the electrodereaction chamber, and the decrease in 02 concentration was

followed over 4 min.ATP. The ATP extraction procedure employed was similar to

ones previously described (19, 20) with some modification for cell

cultures. Two10-ml samples were combined, vacuumfiltered, andweighed. The cells were then frozen in liquid N2 and powderedusing a mortar and pestle. The time fromfiltration to freezing wasnever more than 30 s. The powder was mixed with 7.5 ml of ice-cold, 8% HC104 and allowed to stand on ice for 20 min. Theextracts were transferred to centrifuge tubes and the homogenizerwas rinsed twice with 3.75 ml of 0.1M glycylglycine buffer, pH7.8. The rinses and extracts were combined and centrifuged for 30min at 42,000g and4°C. The supernatants were collected andneutralized to pH 7.8 using 6 N KOH. This liquid was allowed tostand on ice for 15 min to allow complete precipitation of potas-sium perchlorate. The slurry was then centrifuged for 40 min at42,000g and4°C. The supernatant was stored at -70°C untilassayed (generally 4-6 d). Freezing and thawing of samples hadno effect on ATP content (data not shown). Internal standardswere added prior to tissue homogenization.ATP content was measured using the luciferin-luciferase assay

(17). Crystallized and lyophillized luciferase and synthetic, crys-

talline D-luciferin were purchased from Sigma. We used a meas-uring apparatus similar to the one described in Reference 7. Thereaction was initiated by injecting 0.5 ml of ATP standard solutionor diluted extract, and the peak height was recorded. Each day, aworking standard curve in the 10 to 80 pmol range was preparedusing disodium ATP dissolved in water.UV Irradiations. Samples in100-ml beakers were placed 17 cm

beneath two 15 w low pressure Hg vapor lamps andirradiated for10 min with lightfiltered through Permanox plastic. The fluencerate at 254 nm, measured with a thermopile (Eppley), was 2.80 w

E +uv

2.010 -

w

Kx 1.0r V

00z 0

0.0 UV

0 00 200 300 400TIME (MIN)

FIG. 1. Appearance of K+ in the medium of control and iffadiatedcells. Measurements were made by K+-sensitive electrode placed directlyin the cell suspension. The abscissa gives time after the last wash. Datapoints reflect results from three independent experiments. Lines weredrawn by eye. (O) control: no UV. (-), UV ( 1,680 J m-2) given during theperiod indicated.

w

rx

-iL.0z

w

-iLU

1.01

A

v o

I

8 ~~~~~0V

0.5 /

0/0

0.01-10 10 20 30 40

T (OC)FIG. 2. Temperature dependence of UV-induced netK+ efflux. The

ordinate gives the rate at whichK+ appeared in a cell-suspension mediumduring the 60 min following iffadiation (1,680 Jm-2). Each symbolrepresents a separate experiment, except that the point at0°C representsthe mean and SE of four experiments. Each experiment included a control(22°C), and each measurement at other than22°C is expressed relative tothe control rate.

m-2. All samples were agitated continuously during irradiation.Control samples were shielded from the UVlight with Pyrex glass.

RESULTS

UV-Induced Appearance ofKV. Five-day old cultures containedapproximately 5mMK+ in the growth medium, about half that offresh medium. After three washes in 0.01M CaC12, the concentra-tion ofK+ dropped to <0.05mm. Upon incubation, the level of

K+ rose, leveling off at about 0.5mm (0.2-1.0mM) in 2 h (Fig. 1).With further incubation, the cells recovered theK+, reducing theconcentration in the medium to <0.05mm.When washedcells were irradiated with 1,680 Jm-2 at 254 nm,

they lostK+, which appeared in the medium (Fig. 1). There weretwo phases to the appearance ofK+: a more rapid phase that

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UV-STIMULATED K+ EFFLUX

began 10 to 15 min after the UV light was turned on and thatlasted for 30 to 60 min; and a slower phase that lasted for at least16 to 18 h. We never observed any recovery in net K+ by the cells.The rate during the more rapid phase, calculated from 15 inde-pendent experiments conducted at room temperature, was 0.0 16± 0.006 ,umol ml-' min-'. The extent of the loss could be shownby freezing and thawing the cells three times to release all non-bound K+. When this was done, the extemal K+ concentrationrose to 6 mm. Using that value, we could calculate that 3 to 4 hfollowing irradiation, the cells had lost about 30%o of their avail-able, non-bound K+.

Several parameters were tested for their effect on response toUV (data not shown). Number of washings: the response was thesame after one washing as after three. Time after washing: theresponse was the same if the cells were irradiated immediatelyafter washing; the response was slightly faster if the cells wereirradiated 19 h after washing, being detectable 5 min after thebeginning of irradiation. Presence of sucrose: the response was thesame with or without 2% sucrose. Presence of Ca2": the responsewas the same in 1 mm CaCl2 and in 10 mM MgCl2 as in 10 mMCaCl2; when cells were washed with water (no CaCl2) or with

Table I. Influx and Efflux ofK+"Rb+) in Unirradiated and UV-Irradiated Rose Cell Suspensions

Results are means ± SE (number of experiments).

Condition Influx Efflux

pumol m>' min-' x lo3UnirradiatedPrewashed 4.1 ± 0.33 (6) 6.7 ± 4.5 (9)0 h after last wash 0.032 ± 0.028 (4) 0.096 ± 0.038 (4)0.25 h after last wash 0.22 ± 0.015 (2) 0.22 ± 0.22 (2)2.0 h after last wash 0.71 ± 0.18 (6) 0.88 ± 0.32 (6)3.67 h after last wash 0.63 ± 0.13 (9) 0.33 ± 0.20 (8)9.0 h after last wash 2.0 ± 0.79 (3) 0.019 ± 0.019 (4)

UV irradiated 2 h afterlast wash

0.25 h after UV 0.24 ± 0.046 (4) 13.0 ± 2.8 (4)2.0 h after UV 0.88 ± 0.13 (3) 3.8 ± 0.62 (12)

dilute EDTA solution, the net leakage of K+ from unirradiatedcontrols was increased, but the response to UV was greatly de-creased. Temperature (Fig. 2): the response was most rapid at25°C to 30°C; it was completely inhibited at 0°C.

Influx and Efflux. Influx and efflux of 86Rb+ were determinedby short-term incubations with trace amounts of this ion. Forunirradiated cells, the results (Table I) showed that the washingprocedure inhibited both normal influx and efflux. Influx in-creased progressively after the washing, so that it was higher 2and 9 h later. Efflux was higher 2 h, then lower 9 h after washing.In cells that were irradiated 2 h after washing, efflux rose dramat-ically and influx dropped. Both of the effects were greater 15 minafter irradiation than they were 2 h after irradiation. The partialrecovery of normal influx and efflux 2 h after irradiation confirmsthe suggestion above that there were two phases to the leakageprocess.

Assuming that Rb+ was a reasonable analog of K+ for thetransport processes we measured, the results indicated that the netloss of K+ was caused primarily, though not exclusively, by thechange in efflux. The assumption may depend on the transportmechanism under consideration. It seemed to be warranted forUV-irradiated cells, because the difference between the efflux andinflux ofK+ calculated under this assumption (0.01 1 0.002 ,umolml-' min-) was not significantly different from the maximum netrate of appearance ofK+ in the medium (0.0 16 ± 0.006 ,umol ml-'min-'). However, it did not apply so well to the unirradiated cells

after washing; in this case, net leakage was faster than fluxmeasurements would suggest.

Countenons. Though Wright and Murphy (23) established thatthe leakage of K+ induced by relatively low doses of UV is a

specific rather than general membrane effect, during K+ leakagethere must be a balancing flow of another ion to preserve electro-neutrality of the cells. Either a cation enters-in the washed cells,the choices are Ca2' and H+-or an anion exits. To identify thecounterion, we analyzed the filtrate collected from cell suspensionsafter irradiation.

Figure 3 shows a titration of filtrates from irradiated and controlcells. There was a single weak base released from irradiated cells,one with a pKb of 6.4. This matched the pKb of HCO3 and didnot match those of common organic anions. The apparent release

pH

0 10 20

ACID ADDED (1MOL)FIG. 3. Titration of filtrates from control and irradiated cell suspen-

sions. Suspensions were incubated for 2¼4 h; then one was irradiated (1,680J m-2); then they were incubated further for 3¼4 h. Volumes of the filtratesaveraged 7.4 ml. The controls included 7.5 ml of air-saturated 0.01 M

CaCl2 and 7.5 ml of 0.01 M CaCl2 containing 0.01 M KHCO3. Thisexperiment is experiment 2 in Table I.

Table II. Changes in K' and Other Ions after UV Irradiation

Cells were washed with 0.01 M CaCl2 unless otherwise indicated.Analyses were performed on cell-free filtrates. Numbers in parentheses inthe final column give the relative change in HCO3 , C1-, or Ca2 , expressedas a percent of the change in K+ in the same experiment.Experi- Time No UV A

No Ument afterUV UV UV A UV U

min mM

K+ HCO3-1 2.0 0.49 1.90 1.41 0.40 1.53 1.13 (80)2 3.25 0.05 1.51 1.46 0.54 1.82 1.28 (88)

K+ Cl-3a 1.0 0.40 0.99 0.59 0.09 0.09 0.0 (0)4a 1.5 0.61 2.07 1.46 0.16 0.35 0.19 (13)5h 3.25 0.31 1.60 1.29 2.20 2.35 0.15 (12)6a 4.5 0.39 2.05 1.66 0.12 0.14 0.02 (1)

K+ Ca2+7h 1.5 0.25 1.52 1.27 1.03 1.08 0.05 (4)8 3.0 0.07 2.08 2.01 9.95 10.37 0.42 (21)9h 3.25 0.31 1.60 1.29 0.99 0.89 -0.10 (-8)

a Cells washed with 0.01 M CaSO4.bCells washed with 0.001 M CaC12-

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MURPHY AND WILSON

x I

-J E

I-U-wj E.028NcEO :LU-

o- I I

50TIME (MIN)

100

FIG. 4. Effect ofUV on CO2 production by rose cells. The curve showsthe concentration of CO2 in the effluent gas passing over 10 ml ofsuspension culture in a representative experiment. Flow rate was 0.35 mlmin-'. UV dose was 1,680 J m2.

of bicarbonate accounted for approximately 80o to 90%Yo of coun-terion flow (Table II). There was also a small difference in Cl-,accounting for 12% of counterions, in two experiments out of four(Table II), but no difference (<0.01 mM) in N03- or P043-between control and irradiated filtrates. HPLC analysis for organicanions gave a single, unidentifiable peak that was lower in filtratefrom irradiated cells and thus could not represent a counterion toK+ flow.

Irradiation caused a consistent increase in the pH of the cellsuspensions. Normal suspensions adjusted the pH of the mediumto 6.8; after irradiation, the pH of the medium shifted to 7.3 over30 to 120 min. This represented only a small change in H+concentration (about 10-4 mM) and could be explained by theappearance of KHCO3 in the medium.

Irradiation also caused a variable change ofCa2" in the medium.However, a loss was detected in only one experiment out of three(Table II) and accounted for, at most, 16% of the counterion flow.The variability in these data suggests that Ca2" plays no majorrole in the UV response.

Respiration. Measurements of the respiratory rates of controlcell suspensions gave a Qo2 of 0.025 ± 0.0016 ,umol ml-' min-'and a Qco2 of 0.023 ± 0.0023 ,umol ml-' min-'. These two valuesare not significantly different, consistent with the expectation thata culture growing on sucrose should have a respiratory quotientof 1. It is clear that respiration can provide sufficient CO2 tobalance the most rapid UV-induced net efflux of K+.

Irradiation of cells did not change the respiration rate as meas-ured by 02 consumption. The values of Qo215 min and 2 h afterirradiation were 0.026 and 0.023 ,umol ml- min-', respectively.Given the low dose of UV used, these data were consistent withthose reported for tobacco cells (12). Irradiation did reduce theoutput of CO2 (Fig. 4), in accord with the idea that the UVtreatment caused the cells to release KHCO3 instead of CO2.However, the decrease in Qco2 was only 0.087 ± 0.0023 ,umol ml-'min-' (seven determinations), approximately 55% of the mostrapid net K+ efflux.

Effects of Inhibitors. The strong effect of temperature on UV-induced K+ leakage (Fig. 2) suggested that efflux depended onrespiratory metabolism. To test this hypothesis, irradiations wereperformed in the presence of inhibitors. Giving 10-5 M CCCPl 15min before irradiation strongly inhibited UV-induced K+ leakage(Fig. 5), though 1O-6 M CCCP had no effect. CCCP alone, withoutirradiation, caused a slow leakage of K+, but it was not as rapidas that caused by UV. KCN (0.05 mM) and SHAM (0.75 mM) hadan effect similar to that of CCCP at 10-5 M (data not shown).

Neither 10-5 M CCCP, 10-6 M CCCP, nor 0.05 mM KCN plus

'Abbreviations: CCCP, carbonyl cyanide m-chlorophenyl hydrazone;SHAM, salicylhydroxamic acid; DCCD, N,N'-dicyclohexylcarbodiimide;NEM, N-ethylmaleimide; EK, membrane electrical potential due to potas-sium diffusion.

IOO 200TIME (MIN)

FIG. 5. Appearance of K+ in the medium of control and irradiatedcells treated with 10-' M CCCP. (0), no CCCP, no UV; (@), no CCCP,UV irradiated at 120 min; (O), CCCP added at 100 min, no UV; (U),CCCP added at 100 min, UV irradiated at 120 min.

Table III. A TP Content ofRose Cells

Treatment % of Control"5 min 10-6 M CCCP 54

45 min IO-6M CCCP 995 min 10-5M CCCP 41

45 min 10-5 M CCCP 415 min KCN-SHAMa 37

a 0.05 mM KCN plus 0.75 mM SHAM.h Among different experiments, control values varied from 161 to 299

(mean 206) nmol ATP/g fresh wt.

0.75 mm SHAM limited the supply of bicarbonate by reducingQco2. All treatments caused a rise in Qco2 detectable within 1 min;the rise was followed by a recovery to the pretreatment rate over10 to 20 min. We assume that the rise represents a classical Pasteureffect, but we cannot explain the recovery. Direct measurementsof ATP concentration in control and CCCP-treated cells showedthat both 10-6 and 10-5 M CCCP caused an immediate (5 min)drop to about 50% to 40%o of the normal ATP supply, respectively.However, in 10-6 M CCCP the ATP levels recovered within 45min; in 10-5 M CCCP there was no recovery at that time. KCN/SHAM produced an ATP drop similar to 10-5 M CCCP (TableIII). The data strongly suggest that the UV-induced efflux of K+requires normal levels of ATP.We also tested the effects of DCCD (5 x 10-5 and 5 x 10-4 M)

and sodium vanadate (I0O- M), inhibitors ofthe plasma membraneK+-stimulated ATPase. These inhibitors, like CCCP and KCN/SHAM, caused K+ to leak slowly from unirradiated cells. NeitherDCCD nor vanadate stimulated a rapid leakage like that of UV.DCCD strongly inhibited the leakage caused by UV, and vanadateinhibited the later, but not the earlier, phase of leakage caused byUV.

DISCUSSION

A low fluence of UV light does not seem to affect the generalintegrity of higher plant membranes. Evidence for this assertioncomes from studies of tobacco suspension cells, which whenirradiated do not admit mannitol or allow pre-loaded 2-deoxyglu-cose to leak out (23), do not allow detectable amounts of com-pounds which absorb light at 280 nm to leak out, and do notchange their permeabilities to thiourea (22). Rose cells, whenirradiated, do not leak significant amounts of nitrate, phosphate,or calcium ions and leak very little chloride ion (Table II). Thus,the net movement ofK+ out of cells that have been UV-irradiatedmust be interpreted in terms of more specific transport processes.

Because a fraction of the plasma membrane-associated, K+-

UV

I

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UV-STIMULATED K+ EFFLUX

stimulated ATPase is very sensitive to UV radiation (24), it ispossible that the UV-stimulated K+ efflux results from damage tothis enzyme. However, treatments with DCCD and sodium van-adate, which inactivate the ATPase (14), and treatments withCCCP and KCN plus SHAM, which lower the intracellularconcentration of ATP (Table III), did not stimulate a rapid loss ofK+ from the cells as UV did. Thus, if UV stimulates K+ effluxthrough its action on the ATPase, this action must be qualitativelydifferent from the effects of the other agents.

Lichtner et al. (10) have noted an increased efflux of K+ inChara cells treated with NEM. In such cells, and in Chara cellsexposed to 10 mM KCI (8) a great decrease in electrical resistanceof the membrane and the adjustment of the membrane potentialtoward EK suggest an opening of K+-specific channels. While thiswould provide a convenient explanation for K+ efflux in UV-treated rose cells, it cannot be applied without qualification. Anopening of channels should lead to an increase in influx of 'Rb4.Lichtner et al. (10) actually observed such an increase, while wesaw a decrease (Table I). In addition, Keifer and Lucas (8)observed a depolarization when NaCl was added to the externalmedium around NEM-treated Chara cells; we did not see anyeffect of NaCl on K4 leakage from UV-treated rose cells (data notshown). Finally, Ca2" promoted the closing of K4 channels inChara cells transferred from 10 to 0.2 mm KCI (8); in our system,Ca24 promoted K4 leakage. Thus, there were distinct differencesbetween NEM-treated Chara and UV-treated rose cells.The movement of K4 from irradiated cells to the external

medium is accompanied by an appearance of HC03- in themedium. There are several possible ways the flows of K4 andHC03- could be connected. (a) KHCO3 may be transported as aunit. (b) K4 and HCO3 may be transported separately, theirelectrogenic flows connected by the potential difference of theplasma membrane. (c) K4 efflux may be linked to H4 influx, withH4 in the external medium being resupplied by dissociation ofH2CO3 formed from respiratory CO2. We have only fragmentaryclues with which to choose among these possibilities. To distin-guish between the possibilities with certainty, it will be necessaryto study the electrical states of the cell membranes before andafter irradiation.The inhibition of UV-induced K4 leakage by inhibitors of ATP

synthesis suggests that the leakage is an active process. However,there is no other reason to postulate an active mechanism. Thechemical potential of K4 in the washed cells, estimated from theconcentration of K4 in the medium before and after cell lysis andfrom an assumed protoplast volume of 0.06 ml/ml of suspension,was equivalent to a EK of -142 mv, possibly strong enough tooverpower the membrane potential, which for cultured tobaccocells has been estimated to be about -50 mv (2). One alternativeexplanation for the effect of ATP synthesis inhibitors is that theUV-activated transport mechanism for K4 (or HCO3 , or both) isallosterically regulated by the concentration of ATP or a relatedcompound in the cell. Another possible explanation arises fromthe work of Komor et al. (9), who showed that respiratory inhib-itors decreased the efflux of solutes from detergent-treated Chlo-rella, Escherichia, and Saccharomyces cells. They suggested thatlow energy charge changed the structure of the cell membrane sothat it was less sensitive to the treatment.The specificity of K4 efflux suggests that the UV effect and/or

the transporter has a function in the normal life of a plant cell.There have been reports that intermediate-UV radiation (280-310

nm) is necessary for certain developmental responses in plants (4,21), but we have not yet tested the efficacy of these wavelengthsin promoting the K4 efflux response. It is possible that, while thetransporter has a useful function to the cell, the response to UV ispathological, representing a loss of the normal control of thatfunction. Conceivable functions for the transporter include regu-lating concentrations of K+, HCO3 , or H+ inside the cell.

Acknowledgments-We thank Mary Brendler and Peter Matsuura for technicalassistance; Dr. R. W. Rains, R. C. Huffaker, and S. L. Morrison for advice andequipment for HPLC; Dr. Lars Anderson (United States Department of Agriculture)for the atomic absorption spectrometry; and Dr. R. M. Spanswick (Cornell University)for reading and criticizing the manuscript.

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