Plant Physiol. (1985) 79, 207-211 0032-0889/85/79/0207/05/$0 1.00/0
Displacement of Ca2' by Na+ from the Plasmalemma of Root Cells' 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. POLITO Departments ofLand, Air, and Water Resources (G.R.C., A.L.) and Pomology (V.S.P.), University of California, Davis, California 95616 reduced by salinity, but were restored to adequate levels by an additional supply of Ca2" (16). This might indicate that Na+ A microfluorometric assay using chlorotetracycline (CTC) as a probe displaced Ca2" from membranes, inducing K+ to leak out of the for membrane-associated Ca2l in intact cotton (Gossypium hirsutum L. cytoplasm across the plasmalemma. cv Acala SJ-2) root hairs indicated displacement of Ca2 by Na' from Recently, a method was developed to measure chilling-induced membrane sites with increasing levels of NaCl (0 to 250 millimolar). displacement of Ca2" from membrane sites using CTC2 as a K+(MRb) efflux increased dramatically at high salinity. An increase in quantitative, microfluorometric probe for membrane-associated external Ca2+ concentration (10 millimolar) mitigated both responses. Ca2+ (25, 31). CTC, a Ca2+-chelating antibiotic, fluoresces when Other cations and mannitol, which did not affect Ca2+-CTC chelation complexed with divalent cations (5). In an apolar environment properties, were found to have no effect on Ca2`-CTC fluorescence, the configuration of the Ca2+-CTC complex changes, as shown indicating a Na -specific effect. Reduction of Ca2+_C1C fluorescence by by a 5-fold increase in fluorescence emission intensity, while at ethyleneglycol-bis4W-aminoethyl ether) N,N'-tetraacetic acid, which physiological pH the fluorescence characteristics of the Mg2+does not cross membranes, provided an indication that reduction by Na+ CTC complex are similar in polar and apolar surroundings (10, of Ca2+-CIC fluorescence may be occurring primarily at the plasma- 13). In addition, the excitation and emission spectra of the Mg2" lemma. The findings support prior proposals that Ca2+ protects mem- and Ca2+ complexes are sufficiently different so that with optical branes from adverse effects of Na thereby maintaining membrane integ- filters selective for Ca2+-CTC fluorescence changes in the fluority and minimizing leakage of cytosolic K+. rescence signal may be considered indicative of changes in membrane-associated Ca2` (6, 30). For the study described here, this method was adapted to measure changes in membrane-associated Ca2` levels of intact cotton root hairs in response to NaCl treatments at high and low (adequate) external Ca2` concentrations. In this paper, we show that one of the primary responses to salinity in cells of cotton Salt-affected soils make up a substantial portion of the world's roots is the displacement of membrane-associated Ca2` by Na+ land area including approximately 33% of the irrigated soils (9). ultimately leading to a disruption of membrane integrity and Efforts have been made to control salinity by technological selectivity as measured by K+(86Rb) leakage from the root cells. means: reclamation, drainage, use of high leaching fractions, and application of soil amendments. An additional approach to the MATERIALS AND METHODS problem is to utilize the large genetic potential of many crop Plant Material. Cotton (Gossypium hirsutum L. cv Acala SJspecies and select more salt-tolerant crops (3, 9). Selection and breeding for salt tolerance in a particular crop species would be 2) seeds were germinated in the dark at 25°C in a VWR 2020 facilitated if we could identify and understand the mechanisms controlled-temperature incubator. Seeds were placed in germination paper lining a 1000-ml Pyrex beaker and filled with just of salt tolerance and toxicity for that species. Calcium is an important factor in maintenance of membrane enough of a 0.1 modified Hoagland solution (unless otherwise integrity and ion-transport regulation. It has been shown that specified) containing 0.4 mM Ca2" to fully wet the germination Ca2" is essential for K+/Na+ selectivity and membrane integrity paper. This Ca2` concentration is adequate for root growth of (8, 14, 18). Elevated Ca2" concentrations in the nutrient solution cotton under nonsaline conditions (16). Determination of Membrane-Associated Calcium. Ca2+-CTC mitigated the adverse effects of NaCl on bean plants by inhibition of Na+ uptake (12, 17) and reduction in leakiness of membranes fluorescence of individual cells was measured using a microscope (21). LaHaye and Epstein (17) clearly postulated that the Ca2+/ fluorometer equipped with epifluorescence optics and a voltageNa+ interaction takes place at the plasmalemma. These authors stabilized 100-w DC HBO mercury lamp (Carl Zeiss, Inc., NY). suggested that Na+ acted by displacing Ca2+ from membranes, Excitation wavelengths were isolated with a 405-nm bandpass leading to increased membrane permeability and intracellular (9.2-nm bandwidth) interference filter; Ca2+-CTC emissions were isolated with a 530-nm (8.5-nm bandwidth) interference filter Na+ concentrations. Additions of Ca2+ salts to a complete nutrient solution partly (Ditric Optics, Inc., Hudson, MA). Emissions were quantitatively alleviated the suppression of root growth in cotton under high measured using a microcomputer-interfaced Hamamatsu R928 salinity levels (11, 16). Potassium concentrations in the root were photomultiplier. ABSTRACI
2 Abbreviation: CTC, chlorotetracycline.
'Supported by National Science Foundation grant DMB84-04442. 207
CRAMER ET AL.
Plant experiments were performed using intact cotton root hairs of 8-d-old seedlings. A 2-cm segment from the oldest portion of the root having root hairs was placed in a control solution within a custom-built, deep-well microscope slide. The control treatment solution consisted of0.4 mm Ca2", 50gM CTC, and 2.5 mm T-M (pH 6.5) for all plant experiments unless otherwise specified. This solution was removed after the fluorescence determination and replaced with a solution containing the same reagents as the control, plus the treatment reagent. Cutting of the tissue had no effect on Ca2+-CTC fluorescence. The deep-well slide was fabricated from 1.3-cm-thick plexiglass. Solutions and root segments were held in a groove cut in the slide and covered with a glass cover slip. No fluorescence emissions were detected from the plexiglass slide under experimental conditions. The microscope fluorometer was focused on individual root hairs. Background fluorescence was influenced by neighboring root hair cells. This required measurements to be made at the tips of root hairs to minimize this influence. Care was taken to refocus in the same location of the root hair after the treatments were administered. Background fluorescence was subtracted from all measurements. Salt Effect on Ca2-CTC Chelation. A diminished fluorescence could represent either a decrease in the quantity of membraneassociated Ca2" or a change of the chelating properties of CTC. Since Ca2+-CTC fluorescence in 80% (v/v) methanol has the same fluorescence maximum (530 nm) as membranes, it was thought that the properties of Ca2`-CTC chelation in this solution were representative of membranes. The effects on fluorescence of various concentrations of Nae, Ca2", and CTC in 80% methanol were measured with a Perkin-Elmer MPF-44A fluorescence spectrophotometer; the excitation and emission wavelengths were set at 390 ± 10 and 530 ± 5 nm, respectively. The effects on Ca2`-CTC fluorescence of other salts in 80% methanol were measured with the microscope fluorometer as described above. Cellular Location of Ca2-CTC Fluorescence. To assess the contribution of the cell wall to Ca2+-CTC fluorescence, a root segment was treated with 500 mm mannitol. This was sufficient to separate the protoplast from the cell wall in the tips of root hairs, enabling separate measurements of each of these regions. Treatments with EGTA, a Ca2+-specific chelate, were used to remove Ca2' from the extracellular space, including the external surface of the plasmalemma. Ca2+ was left out of the treatment solution, but was included in the control solution prior to exposure of the root to EGTA. Cation Effects on Ca2-CTC Fluorescence. Fluorescence of root hairs was measured after exposure to treatment solutions containing various concentrations of NaCl (O to 250 mM) and CaCI2 (0.4 or 10 mM). The effects of other cations and mannitol were also examined. KI Efflux. Cotton seeds were germinated in a growth chamber under the following conditions: 15 h d; photon flux density of 200 ME m 2 sl; day:night temperature cycle of 25:20C. Seeds were germinated in a 0.1 modified Hoagland solution lacking the KNO3 aliquot. Five-d-old seedlings were transferred to the laboratory (23°C, 15-h d with a photon flux density of 400 ME m-2 s' ) and preloaded with a 0.1 modified Hoagland solution containing 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 to remove residual MRb before placement in the efflux solution. Leakage: of K+(Rb) from intact roots was collected in 15-ml polystyrene centrifuge tubes containing nonlabeled treatment solutions at 23C consisting of various levels of Naa (O to 250 mM) in a 0.1 modified Hoagland solution (minus the KNO3 aliquot). For the purposes of calculating efflux, the lack of K+ in the efflux solution enabled us to assume that K+ uptake was minimal and therefore the internal
Plant Physiol. Vol. 79, 1985
remained relatively constant. The amount of K+('Rb) collected in the centrifuge tubes over various time intervals was determined by Cerenkov counting in a Packard Tri-Carb 300C liquid scintillation system. Semilogarithmic plots oftissue K+(MRb) content versus time were depicted by a triphasic curve (data not shown). The curve generated was similar in characteristics to those observed by other researchers (27). The three phases are interpreted to represent efflux from three cells compartments arranged in series: cell wall, cytoplasm, and vacuole (27). Samples were collected over a 0-min interval in the middle of the second phase. The second phase occurred from 3 to 30 min after initiation of tracer efflux and was interpreted to represent efflux from the cytoplasm through the plasmalemma. Chemicals. 86Rb was obtained from New England Nuclear. All other chemicals were obtained from Sigma.
RESULTS Salt Effect on Ca2-CTC Chelation. The effect of 150 mm NaCl on Ca2-CTC fluorescence in 80% methanol at various 100
10CC() 5 u
°,5 ;19 I
15S0 mM. NaCI (0,0, t---
-). The size
of the 95% confidence interval bars are no longer than the symbols except for the 10
CTC treatment as indicated.
Ca2+-CTC Fluorescence of an 80% Methanol Solution 50OMM CTC, 0.4 mMt CaCI2, 2.5 mM Tris-Mes (pH 6.5), and
Various Salts at a Concentration of 50 mM
Treatment Control (no salt) NaCl
NaNO3 LiCI3 NH4CI
KCl RbCl CsC1 Control (no salt) BaCl2
SrCl2 MgCl2 MnCl2 CuC12 ZnCl2 Pb (NO3)2 LaCI3 I
Mean ± 95% confidence interval.
Ca2--CTC Fluorescence 54.9 ± 3.9a 52.5 ± 3.6 54.6 ± 2.9 58.3 ± 2.7 52.7 ± 4.6 56.4 ± 4.2 55.7 ± 1.8 58.4 ± 3.8
59.5 ± 3.0 64.1 ± 5.0 74.1 ± 4.3 l 16.9 (off scale) 36.0 ± 0.6 25.5± 1.1 r 1 16.9 (off scale) 83.7 ± 11.3 ¢1 16.9 (off scale)
DISPLACEMENT OF Ca2+ BY Na+ FROM THE PLASMALEMMA OF ROOT CELLS Ca2- and CTC concentrations is illustrated in Figure 1. Sodium did not affect Ca2-CTC fluorescence except at Ca2+ concentrations below 0.4 mm. The Ca2+-CTC fluorescence was also dependent upon the Ca24 concentration, but only up to 0.4 mM. The concentration of CTC influenced fluorescence intensity, but did not signficantly alter the Na+-Ca2+ interaction. To test the ion-specifity of Ca2` displacement from membranes by various salts as measured by Ca2+-CTC fluorescence it must be verified that these salts do not affect Ca2+-CTC fluorescence as measured in 80% methanol. The effects of various salts on the Ca2+-chelation properties of CTC in 80% methanol can be seen in Table I. No monovalent cations had any effect on Ca2+CTC fluorescence, but di- and trivalent cations at 50 mm affected fluorescence (except for Ba2+). Cellular Location of [email protected]
Fluorescence. Fluorescence measurements of the protoplast and cell wall of root hairs plasmolyzed in 500 mm mannitol showed that the contribution from the cell wall was less than 5% (data not shown). However, this does not represent a quantitative value for Ca2+ in the cell wall. The polarity in the cell wall is different from that of the membrane; thus, in the cell wall, CTC has a different affinity for Ca2` and Ca2+-CTC has a different wavelength for maximum fluorescence. It can be concluded that Ca2`-CTC fluorescence was principally from membrane-associated Ca2+. Treatments with EGTA reduced Ca24-CTC fluorescence of root hairs (Fig. 2). A large reduction in fluorescence occurred between 10-7 and 10' M EGTA. Further reductions in fluorescence occurred as the EGTA concentration was increased, but to a lesser degree. No cyclosis was observed in root hairs that had been treated with EGTA above 10-' M. Cation Effects on Ca2+-CTC Fluorescence. Figure 3 shows that with 0.4 mm Ca2` increasing concentrations of NaCl progressively reduced the intensity of Ca2+-CTC fluorescence. Attempts to restore Ca2+-CTC fluorescence by replacing NaCl with 50 mM CaC12 failed to increase fluorescence intensity upon return to a control solution. When the roots were exposed to NaCl in the presence of 10 mM Ca2+, reduction of Ca2`-CTC fluorescence occurred only at NaCl concentrations exceeding 25 mm and to a lesser extent than at the lower Ca2l treatment. It should be noted that the data in Figure 3 only represent a relative comparison between the low and high calcium treatments. Fluorescence for the high calcium treatment was greater than the low calcium treatment. In addition to the results of the fluorescence measurements, it was observed that with 0.4 mM CaCl2, cyclosis was inhibited at 150 mm NaCl, but was still visible in solutions
2120 o 400 -_
120 0 100
IS0 200 300 2S0 NoCI (mM) FIG. 3. Effect of increasing levels of NaCl on Ca2e-CTC fluorescence (a quantitative measure of membrane-associated Cal2) in intact cotton root hairs. (O-O), 0.4 mm Ca2"; (0- - .4), 10 mM Ca2l. Each point represents the mean of five replications. The error bars represent 95% 0
confidence intervals for their respective curves.
Table II. Effect of Various Solutes on Ca2'-CTC Fluorescence in Intact Cotton Root Hairs The mannitol concentrations are isosmotic with 50 and 150 mM NaCl, respectively. The external Ca24 concentration was 0.4 mM. Ca2tCTC Fluorescence Treatment % of control
5OmMLiCl 5OmMNaCl 50mMKCI
101±10a 63±6 90±9 102 ± 19 103 ± 16 107 ± 13 101 ± 4 96 ± 9
50 mM RbCl 50 mM CsCI 50 mM BaC12 96 mM mannitol 269 mm mannitol a Mean ± 95% confidence interval.
4QO 0 -
0 U I. 0
...... I'' I ..
....... . .....
6F0 0 IL
0 -i IL U I-U
FIG. 2. Effect of EGTA on Ca2-CTC fluorescence in intact cotton root hairs. The error bar represents the 95% confidence interval for the entire curve.
.. --7 0
FIG. 4. Interaction of Na4 and Ca24 on K+(MRb) efflux rate from the cytoplasm 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 four replications. The error bars represent 95% confidence intervals for their respective curves. -
CRAMER ET AL.
Plant Physiol. Vol. 79, 1985
containing 250 mm NaCI plus 10 mm CaCl2. ionic radius similar to Ca2+ (Table IV). The values for the The effect of those salts which did not affect Ca2+-CTC fluo- hydrated ionic radii are in considerable dispute (2). Different rescence in 80% methanol (Table I) and mannitol on Ca2+-CTC methods of measurement often yield quite different values. Heats fluorescence in intact root hairs can be seen in Table II. Of all of hydration are reflective ofthe degree of hydration and provide the treatments, only Na+ significantly reduced Ca2+-CTC fluo- a better quantitative measurement (2). The point here is that the rescence. results from the displacement of membrane-associated Ca2" corK Efflux. Leakage of K+(86Rb) from higher plant cells can be related well with the crystal ionic radii, but not with the paramused as an index of membrane integrity. The effect of high Ca2" eters of hydration. This suggests that Na+ may displace Ca2` in supply on NaCl-induced membrane leakiness, measured as leak- a site requiring the ions to be in a dehydrated state. This may age from the cytoplasm, is demonstrated in Figure 4. The efflux explain why additions of Ca2` were unable to restore Ca2+-CTC of K+(86Rb) from the cytoplasm began to increase at NaCl fluorescence after root hairs had been exposed to NaCl. If Ca2+ concentrations exceeding 100 mm NaCl. In the low Ca2' treat- were bound to a specific site, such as in a protein, displacement ment, effiux rose sharply with further increases in NaCl concen- of Ca2+ by Na+ could alter the conformation of the binding site, tration. The response in the high Ca2+ treatment was similar up since Na+ has a different charge density than Ca2e. This alteration to 150 mm NaCl, but then diverged dramatically with only a could then prevent access to the binding site by Ca2 . slow rise in K+('Rb) effiux rate. Hauser et al. (15) suggested that the binding of a given cation Ionic effects can be separated from osmotic effects by compar- to negatively charged phospholipid surfaces is primarily deterison of salt to mannitol treatments. Table III shows that efflux mined by the charge density and is largely independent of the in the mannitol treatment that was isosmotic with 225 mm NaCl conformation and hydration of the different polar groups. They was not statistically different from the 225 mM NaCl plus high listed the order of binding for some cations to be: Ca2+ treatment, but significantly lower than the 225 mm NaCI Ca2+ > Ba2 >> Li' > K+ Na+ treatment. Our results do not follow this trend, indicating that the Ca2+CTC fluorescence monitored in our system reflects binding of DISCUSSION membrane-associated Ca2+ to ligands other than those in phosUnder the conditions of this study, Na+ had no effect on the pholipids. properties of Ca2+-CTC chelation. Reduction of Ca2+-CTC fluoEGTA treatments reduced Ca2+-CTC fluorescence (Fig. 2) to rescence by NaCl is interpreted to mean that membrane-associ- about the same extent as NaCl (Fig. 3). EGTA does not cross ated Ca2" was displaced by Na+. Our results showed that NaCl membranes (4, 28), indicating that Na+ displacement of memconcentrations as low as 25 mm reduced the quantity of mem- brane-associated Ca2+ may be occurring primarily at the external brane-associated Ca2+ when the external Ca2' concentration was surface of the plasmalemma. Interestingly, other researchers (23, 0.4 mm. With high external Ca2' supply, root hairs were able to 31) also were unable to eliminate all the Ca2+-CTC fluorescence maintain higher levels of membrane-associated Ca2+ when ex- by EGTA in their systems. This residual fluorescence component posed to high concentrations of NaCl. Other cations did not may represent Ca2+ bound to internal membranes, sites on the reduce Ca2+-CTC fluorescence, indicating that the reduction by plasmalemma with greater affinity for binding Ca2+ than EGTA, Na+ was ion-specific. It may be that other cations can displace or background fluorescence of other di- and trivalent cations that calcium from membranes at higher concentrations than those may affect CTC fluorescence (Table I). tested in this study, or for those cations which could not be tested Table III shows that there was an increase in K+(86Rb) efflux due to their effects on Ca2+-CTC chelation. at high salinity and high calcium relative to the control treatment. Of the cations tested, Na+ is the only cation with a crystal There was a similar increase caused by an isosmotic concentration of mannitol. In addition, mannitol did not affect Ca2+-CTC Table III. Effect of Isosmotic Solutions of NaCl and Mannitol on fluorescence in root hairs (Table II), so that the increase in effiux K+(86Rb) Efflux from the Cytoplasm of Roots of Intact Cotton Seedlings under these conditions was not the result of Ca2+ displacement See "Materials and Methods" for details of nutrient solutions from the plasmalemma. Since K+(86Rb) effiux was not signifiTreatment K+ ('Rb) Efflux cantly different at either the high calcium/salinity or mannitol treatment, the increase in effiux was probably the result of % of control osmotically induced changes in plasmalemma permeability. Ta225 mm NaCl 686 ± 20a ble III also shows that K+(86Rb) effiux at high salinity and low 225 mM NaCI + 10 mm CaC12 300 ± 133 calcium was significantly greater than at high salinity and high 271 ± 149 393 mM mannitol calcium. This additional leakage of K+(86Rb) can be attributed a Mean ± 95% confidence interval. to ion toxicity to the plasmalemma caused by the high salt concentration, which can be mitigated by a higher external Ca2+ Table IV. Physical Chemical Characteristics of Various Cations supply. Although it appears that some displacement of membraneHeat of Hydrated Crystal associated Ca2+ by Na+ did occur in the high Ca2` treatment Cation Ionic Hydration Ion1ic Rai' Iatninradis (Fig. 3), this may not have induced any increase in membrane Radiib (AH)Y leakiness. The increase in membrane leakiness in the high Ca2` nm kcal/mol treatment was most likely due to effects induced by osmotic Li+ 0.068 0.37 -121 stress, as discussed in the previous paragraph. It may be that a Na+ 0.097 0.30 -95 certain degree of Ca2+ displacement from the plasmalemma must K+ 0.133 0.27 -75 be reached before severe changes in permeability occur. This Rb+ 0.147 0.24 -69 conclusion was inferred from experiments in which beet root Cs+ 0.167 -61 tissue was exposed to various concentrations of EDTA (26) and Ba2+ 0.134 -308 could explain the differences in salt-stress response observed Ca2+ 0.099 0.44 -377 between the two Ca2+ treatments. Alternatively, reduction in b a Taken from Ref. 7. c Taken from Ca2`-TC fluorescence by salinity and high calcium may repreEstimated from Ref. 22. Ref. 2. sent displacement of Ca2" by Na+ from membrane sites different
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 speculative. Leakage of K+ may be the direct result of Ca2" displacement by Na+ from the membrane opening potassium channels. Alternatively, other indirect factors may be responsible for this response, such as depolarization of the membrane potential which can result from a rise in intracellular Ca2". In support of the latter hypothesis is the observation that inhibition of cyclosis correlated with the large leakage of K+ in the low calcium treatment, and rises in intracellular Ca2+ concentration have been shown to inhibit cyclosis in Chara (29). Na+ displacement of membrane-associated Ca2" could increase membrane permeability allowing increases in Ca2+ influx, but at high external calcium concentrations this increase in permeability may be minimized. Maintenance of adequate K+ concentrations in root cells is crucial for continued growth. K+ is needed not only for cell turgor to drive cell expansion, but also as a cofactor for many enzymes (20). Furthermore, the leakage of K+ from the cell lowers the K+/Na+ ratio in the tissue (16). K+/Na+ selectivity has been found to be an important factor in the salt tolerance of cotton (19). The data presented in this study strongly indicate that high Na+ concentrations displace Ca2" from the plasmalemma, resulting in a loss of membrane integrity and efflux of cytosolic K+. Young and Kauss (32) attempted to study the effect of polycations, polyamines, and polyanions on cell surface calcium, and their relation to membrane permeability using 45Ca2+ to measure Ca2" displacement. They found that the amount of 45Ca2' displaced from isolated cell wall fragments was similar to whole cells and that permeability did not correlate with 45Ca2' displacement. These effects are difficult to interpret, because it is not clear whether cell wall and/or plasmalemma Ca2` was displaced. Since the cell wall contains the largest proportion of total cell calcium (14), we suggest that a large displacement of Ca2' from the cell wall would mask any displacement from the plasmalemma. Their data showing displacement of 45Ca2' by different cations cannot be compared to ours for the reasons stated above and because they used much lower cation concentrations (0.5 mM). Clearly, our method was capable of distinguishing these two components. However, the Ca2+-CTC fluorescence technique appeared to be unsuitable for studying the effects of most di- and trivalent cations on membrane-associated Ca2+, since these 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 cotton cultivar. Whether or not this holds true for other cultivars or species remains to be verified. Recently, differential growth responses to salinity as affected by external calcium have been observed within a species (1, 24). It would be interesting to know if these differences can be attributed to differences in displacement of membrane-associated Ca2` or to some other factor. In summary, Na+ displaced membrane-associated Ca2+ from cotton root hairs. This response was Na+-specific and may be occurring primarily at the external surface of the plasmalemma. Both the displacement of membrane-associated calcium and the increase in membrane leakiness were mitigated by a high external Ca2" supply. Acknowledgments-We thank Dr. E. Epstein and J. Lynch for critical reading of this manuscript, and the latter for his suggestions and assistance in the efflux studies.
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