dissipation, gradients tonoplast vesicles and liposomes ... · forquin-acrine (final concentration...

Plant Physiol. (1988) 86, 1315-13220032-0889/88186/1315/08/$01.00/0

Dissipation, of pH Gradients in Tonoplast Vesicles and Liposomesby Mixtures of Acridine Orange and AnionsIMPLICATIONS FOR THE USE OF ACRIDINE ORANGE AS A pH PROBE

Received for publication August 24, 1987 and in revised form January 4, 1988

ANDREW J. POPE* AND ROGER A. LEIGHAFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, HertfordshireAL5 2JQ, United Kingdom

ABSTRACT

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast ves-icles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgarisL.) storage tissue. When used as a fluorescent pH probe in the presenceof 1, ClO3-, NO3-, Br-, or SCN-, acridine orange reported lower pHgradients than either quinacrine or [14C]methylamine. Acridine orange,but not quinacrine, reduced ['4C]methylamine accumulation when NO3-was present indicating that the effect was due to a real decrease in thesize of the pH gradient, not a misreporting of the gradient by acridineorange. Other experiments indicated that acridine orange and NO3- in-creased the rate of pH gradient collapse both in tonoplast vesicles and inliposomes of phosphatidylcholine and that the effect in tonoplast vesicleswas greater at 24C than at 12°C. It is suggested that acridine orange andcertain anions increase the permeability of membranes to H+, possiblybecause protonated acridine orange and the anions form a lipophilic ionpair within the vesicle which diffuses across the membrane thus discharg-ing the pH gradient. The results are discussed in relation to the use ofacridine orange as a pH probe. It is concluded that the recently publishedevidence for a NO3-/H+ symport involved in the export of NO3- fromthe vacuole is probably an artefact caused by acridine orange.

Although detailed studies of the transport systems at the ton-oplast are still in their infancy, much progress has been madesince methods were developed for the isolation of intact vacuolesand sealed tonoplast vesicles (20, 29). Studies with vesicles havebeen particularly fruitful and have shown that the tonoplast pos-sesses two inwardly directed, electrogenic H+-pumps, one en-ergized by ATP the other by PPi (e.g. 1, 9, 24, 26, 33). TheApHl (inside acid) and AI (inside positive) generated by thesepumps provide the driving force for secondary transport systemsresponsible for the movement of other solutes across the tono-plast (e.g. 2-4, 8, 18, 25, 27, 28, 30).

Proton-linked secondary transport systems in tonoplast vesi-cles have been studied indirectly by observing the effects of addedsolutes on pH gradients generated with either ATP or PPi (3,27), or created artificially (4, 5, 28). Changes in the vesicle ApHhave been followed either by measuring the accumulation of aradio-labeled weak base such as [14C]methylamine (10), or bymonitoring the change of the fluorescence of pH-sensitive probes

I Abbreviations: ApH, pH gradient; At, membrane potential; BTP,1,3 bis-(tris[hydroxymethyll-methylamino)-propane; IDA, iminodiace-tate; PPase, inorganic pyrophosphatase.

such as quinacrine, acridine orange, or 9-aminoacridine (2). Anessential assumption underlying the use of these probes is thatthey are passive reporters of ApH which do not, themselves,induce changes in the size of the pH gradient formed. Here weshow that this assumption may not be valid for acridine orangewhen NO3- or certain other anions are present. The effects ofmixtures of NO3- and acridine orange on ApH in tonoplast ves-icles have been investigated in detail. The results reconcile dif-ferences in the literature concerning the effects of NO3 on PPi-dependent ApH formation (3, 25). In addition, they lead us toquestion the published fluorescence evidence for the presenceof a NO3-/H+ symport involved in the export of NO3- from thevacuole (3).

MATERIALS AND METHODS

Plant Material and Chemicals. Seeds of oat (Avena sativa L.cv Trafalgar) were washed in running tap water for 2 to 8 h andthen germinated and grown in the dark at 25°C over an aeratedsolution of 0.5 mM CaSO4. Roots were used when the seedlingswere 4 d old. Red beet (Beta vulgaris L. cv Detroit CrimsonGlobe) was grown in pots in a glasshouse and storage roots wereharvested immediately before use.The majority of chemicals were obtained from B.D.H. Chem-

icals Ltd. or from Sigma Chemical Co. (both of Poole, Dorset,U.K.). ATP was from Boehringer Mannheim (Lewes, East Sus-sex, U.K.). Dextran T70 was supplied by Pharmacia (MiltonKeynes, U.K.). [14C]Methylamine (59 mCi/mmol) was purchasedfrom Amersham International, Amersham, Bucks, U.K. Tetra-sodium PPi and disodium ATP were converted to their BTP saltsby cation exchange chromatography with Dowex-50W-X8 resin(H+-form) and titration to pH 7.4 with BTP.

Isolation of Tonoplast Vesicles. Tonoplast vesicles were iso-lated from oat roots as previously described (25). Those fromred beet storage roots were isolated using either the method ofBriskin et al. (7) or that of Rea and Poole (26). The method ofisolation of the beet vesicles did not affect the results obtained.Measurement ofpH Gradient Formation in Tonoplast Vesicles.

Development of an acidic pH within the vesicles was measuredusing both fluorescent and radioactive probes. The quenching ofthe fluorescence of quinacrine or acridine orange at 25°C wasmeasured in a stirred cuvette using a Perkin-Elmer LS5 spectro-fluorimeter (Perkin-Elmer Ltd., Beaconsfield, Buckingham-shire, U.K.) interfaced to an IBM-XT microcomputer. For quin-acrine (final concentration 2 gM) the excitation and emissionwavelengths were 420 and 495 nm, respectively, and the corre-sponding values for acridine orange (final concentration 5 gM)were 495 and 540 nm. The assay medium for PPi-dependent H + -transport contained 250 mm glycerol, 25 mm Hepes-BTP (pH

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Plant Physiol. Vol. 86, 1988

7.4), 0.35 mM EGTA-BTP (pH 7.4), 150 /LM PPi-BTP, and 50mm K-salts in a final volume of 2 ml. In the experiments whereanions were added as their BTP salts, all assays contained 50mM K-IDA (pH 7.4) since the PPase requires K+ for maximalactivity (26, 32, 33). ATP-dependent ApH formation was meas-ured in a medium containing 250 mm glycerol, 25 mM Hepes-BTP (pH 7.4), 0.35 mM EGTA-BTP, 1.5 mM ATP-BTP, andsalts as indicated in the legends. Vesicles (20-50 ,g protein)were preincubated in these media and the reaction was startedby the addition of MgSO4 to a final concentration of 5 mm.Gradients were collapsed by adding gramicidin-D to a final con-centration of 2.5 ,tg ml- l. When the effects of temperature wereinvestigated, the reaction mixtures were preequilibrated at thedesired temperature and the fluorescence cuvette was thermo-stated at that temperature. To prevent condensation, a streamof N2 was directed over the sides of the cuvette (14). At the endof each assay, the temperature in the cuvette was measured witha thermometer. In all experiments, the data output from thefluorimeter was collected and analyzed using software developedby Jennings et al. (17).Uptake of ['4C]methylamine was measured at 25°C in a re-

action mixture (final volume 0.5 ml) consisting of 250 mm glyc-erol, 20 mM Hepes-BTP (pH 7.4), 5 mM MgSO4, and 20 FM['4C]methylamine (1.18 ,uCi ml-l), 300 pM PPi-BTP, and 50 mMK-salts as indicated. Uptake was started by the addition of ves-icles (40-75 ,ug of protein) and was terminated by filtration 5min later when accumulation had reached a steady state (25).Four 100 ,ul aliquots from each treatment were collected onseparate Whatman WCN cellulose nitrate membrane filters (0.45,um pore size) as previously described (25). The filters were driedand the radioactivity determined by liquid scintillation counting.PPi-independent uptake was measured in samples containing 50mM KCI but no PPi.

Preparation of Liposomes. One g of soybean phosphatidyl-choline (Sigma, Type IV-S) was dissolved in 10 ml of chloroformand 100 ,ul aliquots were dispensed into screw-cap glass vials.The chloroform was removed under a stream of N2 at roomtemperature to leave a thin film of phospholipid on the wall ofthe vial. One ml of N2-purged 100 mm citric acid-Na2HPO4 buffer(pH 5.4) was added to the vial, the air was displaced with N2,and the cap was replaced. The mixture was then sonicated at4°C in an ultrasonic bath until no further clarification occurred.The liposomes were rapidly frozen in liquid N2 and thawed im-mediately before use. Liposomes were loaded with 50 mM KCIor KNO3 by including these in the sonication buffer.Measurement of pH Gradients in Liposomes. An acidic pH

gradient of 2 units was imposed by transferring 30 Al of liposomesto 2 ml of 100 mm citric acid-Na2HPO4 (pH 7.4). The resultantApH was measured using either quenching of acridine orangefluorescence or uptake of ['4C]methylamine, essentially as de-scribed for tonoplast vesicles. For assays using liposomes loadedwith KCI or KNO3, the appropriate salt was also included in theexternal medium at a concentration of 50 mm.

Protein Determination. Protein was measured using a dye bind-ing assay (6) with BSA as a standard.

RESULTSIn initial experiments, PPi, rather than ATP, was used as the

energy source for H+-pumping because the tonoplast PPase isinsensitive to anions (25, 26, 32). Thus, effects of anions on PPi-dependent ApH formation were not complicated by direct effectson the H+-pump itself. In contrast, the tonoplast H+-pumpingATPase is stimulated by Cl- and inhibited by NO3- (31). Max-imal rates of PPi-dependent H+-pumping require a PPi concen-tration of about 100 uM; higher concentrations reduce activity(21, 33). The concentrations of PPi employed were as close aspossible to this optimum without causing substantial substrate

depletion. The concentrations of quinacrine (2 gM) and acridineorange (5 FM) were those most commonly used in publishedwork with tonoplast vesicles (2, 3, 9, 25, 26, 33).The Effects of Anions on PPi-Dependent pH Gradient For-

mation Measured using Quinacrine and Acridine Orange. Whenquinacrine was used as a pH probe with tonoplast vesicles fromeither oats or beet, both the initial rate and total extent of flu-orescence quenching, and therefore of ApH formation, weregreatest when measured in the presence of Cl-, Br-, or NO-(Fig. 1, A and B). However, with acridine orange, both the rateand extent of quenching were very much less with NO3- thanwith Cl- or Br- (Fig. 1, C and D). In five experiments with oattonoplast vesicles, the gramicidin-reversible quenching measuredwith acridine orange in the presence of NO3- was 21 ± 3%(mean ± SE) of that with Cl-, whereas with quinacrine it was103 ± 4%.The difference in the response of the two probes was confirmed

when the effect of increasing concentrations of NO3- or Cl- onPPi-dependent ApH formation was examined. With quinacrine,NO3- stimulated the apparent rate of ApH formation in theabsence of Cl-, but had little effect on the high rates measuredin the presence of 50 mm Cl- (Fig. 2A). However, with acridineorange, NO3- was unable to stimulate fluorescence quenchingin the absence of Cl- and progressively prevented stimulation

Quinacrine

V

<K-IDA~A K2S04 1

Br I

KN03 O- A

B

KNONO_ja

Acridine orange

v; CD

A

FIG. 1. Effects of anions on PPi-dependent fluorescence quenchingof quinacrine (A, B) and acridine orange (C, D) by tonoplast vesiclesfrom oat roots (A, C) and red beet storage roots (B, D). Vesicles (ap-proximately 20 jig protein) were preincubated in assay media containingthe salts indicated at a final K+ concentration of 50 mm. Proton transportwas initiated by the addition of MgSO4 to a final concentration of 5 mM(v). Gradients were collapsed by adding gramicidin-D to a final con-centration of 2.5 ,ug ml-l (A). Other conditions were as described in"Materials and Methods."

1316 POPE AND LEIGH

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COLLAPSE OF pH GRADIENTS BY ACRIDINE ORANGE

c 15

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0J C 3 0 5

0

* Bo 250

20

10

5-

0 10 2C 30 40 50

NO3-BTP (mM)

FIG. 2. Response to N03- concentration of PPi-dependent ApH for-mation by tonoplast vesicles from oat roots, measured using either quin-acmne (A) or acridine orange (B) as the pH probe. Assays containedeither 0 (0) or 50 (0) mm C1-BTP. Potassium was supplied as 50 mMK-IDA (pH 7.4). Other conditions were as described in "Materials andMethods."

by 50 mM Cl- (Fig. 2B). When the Cl- concentration was variedin the absence of NO3-, both probes reported a progressivestimulation of H+-pumping (Fig. 3). In contrast, when the Cl-concentration was varied in the presence of 50 mM NO3-, uni-formly high rates of H+-pumping were reported by quinacrineat all Cl- concentrations (Fig. 3A), but no H+-pumping wasmeasurable with acridine orange (Fig. 3B).The effect of NO3- was temperature dependent (Fig. 4). At

12°C the initial rate of PPi-dependent quenching of acridineorange fluorescence was similar with both 50 mM Cl- and NO3-,although the total quench was slightly larger with Cl . However,as the temperature was increased, the size of the quench obtainedwith NO3- was reduced, but that with Cl- was not substantiallyaltered. These changes were not observed with quinacrine (Fig.4). At 25°C, the effect was dependent on acridine orange con-centration and saturated at 5 to 10 ,UM acridine orange (notshown).

Effects of Anions and Acridine Orange on ApH Measured with['4ClMethylamine. As the responses of the two fluorescent probeswere so different, [14C]methylamine accumulation was used asan alternative measure of relative ApH. The effects of differentanions on steady state [14C]methylamine accumulation were com-pared with their effects on the gramicidin-recoverable fluores-cence quenching of both quinacrine and acridine orange. Foreach probe, the results with different anions were expressed asa percentage of the pH formed in the presence of Cl-. Resultsobtained with quinacrine and [14CJmethylamine were generallyin good agreement but acridine orange reported smaller gradientsthan the other probes when I-, NO3-, Br-, and SCN- werepresent (Table I). The largest difference in behavior was ob-

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FIG. 3. Response to Cl- concentration of PPi-dependent ApH fo!-mation by tonoplast vesicles from oat roots, with quinacrine (A) oracridine orange (B) as the pH probe. Assays contained either 0 (0) or50 mm N03-BTP (0). Potassium was supplied as 50 mm K-IDA (pH7.4). Other conditions were as described in "Materials and Methods."

served with I - which was as effective as Cl- at stimulating ApHmeasured with quinacrine, but was totally ineffective when ac-ridine orange was used. However, further experiments focussedon the effects of NO3- because of the physiological importanceof this anion in plants. In addition, the effect of I- appeared tobe different to that ofNO3-, in that quenching of acridine orangefluorescence was observed in the presence of I- (data not shown),but none of the quenching was recoverable with gramicidin (TableI), while with NO3-, the quenching was recovered (e.g. Fig 1C).The comparison of the pH probes suggested that, in the pres-

ence of certain anions, acridine orange either misreported thepH gradient that was formed or interfered with its formation.To distinguish between these two alternatives, acridine orange,and quinacrine were added to [14C]methylamine uptake assaysto see whether they directly affected PPi-dependent ApH for-mation. Methylamine was accumulated to similar levels in bothcontrols and in treatments containing 2 FM quinacrine (TableII). However, adding 5 ILM acridine orange caused an appreciabledecrease in [(4C]methylamine accumulation and this decreasewas larger with NO3- than with Cl1-. Acridine orange also alteredthe response of ['4C]methylamine accumulation to changes inNO3- concentration. In control assays, with no acridine orangepresent, increasing NO3- concentrations progressively stimu-lated ApH formation (Fig. 5) as found when using quinacrine asa pH probe (Fig. 2A). However, when 5 ,UM acridine orangewas present, there was no stimulation of ['4Cjmethylamine ac-cumulation as NO3- concentration was increased, as found whenusing acridine orange as a pH probe (Fig. 2B). These resultsindicated that acridine orange correctly reported the ApH thatwas formed when it was used as a fluorescent pH probe but it

K1

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1317



POPE AND LEIGH

Acridine orange


Table I. Comparison of the Effects of Various K+ Salts on PPi-Dependent ApH Formation by Oat Tonoplast Vesicles, Measured with

Three Different pH ProbesEach probe was used under the conditions described in "Materials

and Methods." Salts were added to a final K+ concentration of 50 mM.Methylamine accumulation was terminated after a 5 min incubation.Gramicidin-recoverable fluorescence quenching was used as the measureof relative ApH with quinacrine and acridine orange. The results are themean of three experiments.

Salt ApH (% KCI)Quinarine AcridineAdded Methylamine Quinacrine rne

KCI 100 100 100KBr 99 102 75KNO3 93 104 21KI 75 103 0K2SO4 21 21 29KSCN 19 15 4K-IDA 15 12 9K-malate 14 9 10

Table II. Effect of 2 gM Quinacrine and 5 um Acridine Orange onPPi-dependent ['4C]Methylamine Accumulation in the Presence

of 50 mnKCl or 50 mM KNO3Results are the mean ± SE of four experiments

['4C]Methylamine Accumulation

Salt Added (% KCI control)

Control + Quinacrine + Acridineorange

KCI 100 96 ± 16 66 ± 8KNO3 110 ± 15 88 ± 17 27 ± 12

FIG. 4. Effect of temperature on PPi-dependent ApH formation bytonoplast vesicles from oat roots with quinacrine (left hand traces) oracridine orange (right hand traces) as the pH probe. Vesicles were prein-cubated in assay media containing 50 mm KCI or KN03 at the temper-ature indicated. Proton-transport was initiated by the addition of MgSO4to a final concentration of 5 mM (V). Gradients were collapsed by addinggramicidin-D to a final concentration of 2.5 ,ug ml-' (A). Other con-ditions were as described in "Materials and Methods."

interacted with NO3- to reduce the size of the gradient.Effects of Anions on ATP-Dependent H+-Transport Measured

with Acridine Orange or Quinacrine. In order to deterniine whetherthe effects of acridine orange were general, we tested whetherpH gradients generated by the tonoplast ATPase were also af-fected. However, using NO3- for these experiments was com-plicated by the direct inhibition of tonoplast ATPase activity bythis anion (31). Thus, NO3- added before the initiation of ATP-dependent H+-pumping inhibited ApH formation, irrespectiveof the pH probe that was used (see also Refs. 1, 10, 24). How-ever, evidence that ATP-generated pH gradients could be af-fected was obtained by adding NO3- after the H+-pump hadbeen activated in the absence of anions. These experiments wereperformed with beet tonoplast vesicles to mimic similar experi-ments which had provided evidence for the presence of a NO3 /H+ symport at the tonoplast (3). Activation of the ATPase bythe addition of Mg2+ in the absence of Cl- or NO3- induced aslow rate of H+-pumping that was greatly stimulated by thesubsequent addition of Cl- or NO3 (Fig. 6; see also Refs. 3,22). With Cl-, the ApH that was formed was relatively stable

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2001

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0 10 20 30 40 50NO3 -BTP (mM)

FIG. 5. Effect of acridine orange on the response to NO3- concen-

tration of PPi-dependent ['4C]methylamine accumulation by tonoplastvesicles from oat roots. Assays contained either 0 (0) or 5 AM (0)acridine orange. All treatments contained 50 mM K-IDA (pH 7.4). Otherconditions were as described in "Materials and Methods."

and similar results were obtained with quinacrine and acridineorange (Fig. 6, A and B). With NO3-, however, the stimulationof ApH was followed by a collapse of the gradient (Fig. 6, Aand B) and the rate of this collapse was much faster with acridineorange than with quinacrine (Fig. 6C).

Additional evidence that ATP-dependent pH gradients werereported differently by acridine orange and quinacrine was ob-tained using oat tonoplast vesicles in the presence of C103 -

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Fid. 6. Effect of adding NO3- or Cl- after the initiation of ATP-dependent pH formation by tonoplast vesicles from red beet storage roots.Proton pumping was initiated at (V) by the addition of MgSO4 to a final concentration of 5 mm. Nitrate-BTP or C1-BTP were added at (V) to givethe final concentration indicated. A, Responses measured using quinacrine as the pH probe; B, responses measured using acridine orange as thepH probe; C, the rate of reversal of fluorescence quenching by NO3- measured with quinacrine (M) or acridine orange (0). In A and B the dashedline indicates the response to 30 mm Cl1-. Gradients were collapsed at ( T ) by adding gramicidin to a final concentration of 2.5 ,ug ml '.

Although this anion has been used as an analog for NO3- intransport studies (12), it is far less inhibitory to the tonoplastATPase than NO3- (16). The effects of C103 were measuredat two different Cl- concentrations to determine whether therate of H+-pumping affected the results obtained. Increasingconcentrations of C103 - had little effect on ATP-dependent H + -pumping when quinacrine was the pH probe, irrespective of theCl- concentration (Fig. 7A). In contrast, with acridine orange,C103- progressively inhibited the H+-pumping measured witheither 10 or 50 mM Cl- (Fig. 7B). The overall decrease in therate of quenching was similar at both Cl- concentrations, but,because the rate of H+-pumping was lower with 10 mM Cl-,C103- caused a greater percentage inhibition at this Cl- con-centration (Fig. 7C).

Effects of Acridine Orange on H+-Leakage from TonoplastVesicles and Liposomes. The reduction of both PPi- and ATP-dependent ApH by acridine orange and NO3- suggested a gen-eral effect on H+ permeability and this was investigated by de-termining whether mixtures of acridine orange and NO3- in-creased rates ofH + leakage from tonoplast vesicles and liposomes.In the experiments with tonoplast vesicles, a ApH was generatedwith PPi and then the residual PPi was hydrolyzed by addingsoluble PPase from yeast. Addition of the soluble PPase col-lapsed the ApH after a lag that was dependent on the amountof PPase added. To determine the effects of Cl - or NO3- onthe rate of collapse, the amount of PPase added was adjustedso that the lag was about 25 s and the anions were added duringthis period. With quinacrine, neither anion affected the rate ofcollapse, whereas with acridine orange the rate of collapse in-creased with NO3-, but not Cl-, concentration (Fig. 8).For the experiments with phosphatidylcholine liposomes, a

ApH of 2 units was imposed by adding lipopsomes containingbuffer at pH 5.4 to a stirred medium buffered at pH 7.4 andcontaining 5 ,UM acridine orange. The addition of the liposomescaused a quenching of the fluorescence of the acridine orangeas it accumulated in the acidic interior of the liposomes (11, 19).With liposomes prepared in buffer without KCl or KNO3, theApH was relatively stable, and the presence of 50 mm KCl orKNO3 in the medium to which they were added had no effecton the rate of collapse of the gradient (Fig. 9A). When liposomescontained 50 mm KCl or 50 mm KNO3, the ApH was less stable

but collapsed much more rapidly with the liposomes containingKNO3 (Fig. 9B). Nine separate measurements made on threedifferent liposome preparations yielded a mean ± SE rate offluorescence quench reversal for KNO3-loaded liposomes of 32.7± 2.7% min-' but only 6.8 + 3.4% min-' for KCl-loadedliposomes and 2.5 + 0.4% min-' for unloaded liposomes. Toensure that the presence of NO3- during the preparation of theliposomes had neither interfered with the formation of the li-posomes nor reduced liposome integrity, ['4C]methylamine ac-cumulation was used to test independently the stability of theimposedApH. Both the time course and extent of [14C]methylamineaccumulation were unaffected by the presence of KCI and KNO3within the liposomes (Fig. 10), indicating that the permeabilityproperties of the liposomes were unaffected by loading. How-ever, when 5 /LM acridine orange was present in the externalmedium it reduced [14C]methylamine accumulation and this re-duction was much larger with liposomes containing KNO3 (Fig.10). Thus the instability of the pH gradients was directly causedby acridine orange and was not the result of loading KNO3 intothe liposomes.

DISCUSSIONThe results presented in this paper show that the responses to

anions of dpH formation in tonoplast vesicles is altered by ac-ridine orange. When used as a fluorescent pH probe in the pres-ence of I-, C103 -, NO3-, Br-, or SCN-, acridine orange re-ported much smaller pH gradients than either quinacrine or[14C]methylamine (Table I). This was because mixtures of acri-dine orange and these anions changed the permeability of mem-branes to H+, thereby enhancing leakage of H + and dissipatingApH. Although we observed the largest responses with I-, NO3-and C103-, it is probable that acridine orange has an effect ondpH irrespective of the anion that is present, because[14C]methylamine accumulation by tonoplast vesicles and lipo-somes was reduced by it under all conditions tested (Table II;Fig. 10). It therefore seems that acridine orange cannot be con-sidered to be a passive reporter of pH gradients under any con-ditions, since it is always likely to reduce pH. The mechanismby which this increased H+ leakage is induced is unclear, butthe liposome experiments (Figs. 9 and 10) indicate that it requiresthe anions to be within the vesicles. One possibility is that the

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FIG. 7. Response of ATP-dependent ApH formation by oat tonoplastvesicles to increasing concentrations of C103 - in the presence of 10 (opensymbols) or 50 (closed symbols) mm Cl-. A, ApH measured with quin-acrine; B, ApH measured with acridine orange; C, activity as percentageof control, measured with quinacrine (A, A) or acridine orange (0, *).

internal anions and acridine orange cause a loss of membraneintegrity. Alternatively, the mechanism might be more subtlesuch as an interaction of the anions with protonated acridineorange within the vesicle to form a lipophilic ion pair that is ableto diffuse out of the vesicle and thus effectively transport a protonback to the external medium. Such a mechanism has been pro-posed to account for the uncoupling effect of amine local an-esthetics in mitochondria (15). The monoamine, acridine orange,probably forms an ion pair more readily than the diamine quin-acrine. In addition, the temperature dependence of the effectsof acridine orange (Fig. 4) suggests that membrane fluidity maybe a factor determining the size of the leak that is induced.The extent to which pH gradients in membranes other than

tonoplast will be affected by acridine orange remains unclear.Effects similar to those reported here were observed in submi-tochondrial particles from beef heart (13) but not in plasmamembrane vesicles from Neurospora (23). Thus, the effects mightbe limited to certain types of membrane although the basis forsuch a specificity remains unclear. Despite this, it would seemprudent to assume that pH gradients in any membrane could bedissipated by acridine orange when anions are present and to

rE50 -

40

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'a 20 _-

0 10 20 30 40 50Anion-BTP (mM)

FIG. 8. Effect of N03 (E0. 0) and Cl- (U, 0) concentration on therate of passive collapse of pH gradients in oat tonoplast vesicles, meas-ured using quinacrine (0l, U) or acridine orange (0,0). Proton pumpingwas initiated by the addition of Mg2+ in the presence of 50 mM KCI toensure maximal pH gradient formation. Four min later, after a steadystate ApH had been achieved, 9 units of soluble PPase from yeast wereadded, followed by N03-BTP or Cl-BTP to give the final concentrationindicated. In each case the dilution factor upon adding anions was keptconstant. The time course of ApH collapse was fitted to a rising expo-nential function using software developed by Jennings et al. (17) and theinitial rate of fluorescence reversal was calculated from this function.

AV

L aB

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2min

FIG. 9. Quenching of acridine orange fluorescence induced by im-posing aApH of 2 units (inside acid) on liposomes of phosphatidylcholineprepared in 100 mm citrate phosphate buffer (pH 5.4). Thirty ,1l ofliposomes were added at (V) to 2 ml of 100 mm citrate-phosphate buffer(pH 7.4), and residual ApH was collapsed at (A) with 0.015% (w/v)Triton X-100. A, Liposomes containing buffer only were added to amedium containing either a, no salts; b, 50 mm KCl; or c, 50 mm KNO3.B, Both liposomes and medium contained either, a, 50 mm KNO3, orb, 50 mM KCI.

1320 POPE AND LEIGH




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FIG. 10. Effect of acridine orange on the accumulation of[14C]methylamine by liposomes of phosphatidylcholine after the impo-sition of a ApH of 2 units (inside acid). The liposomes were loaded with100 mm citrate-phosphate buffer (pH 5.4) containing A, no salts; B, 50mM KCI; or C, 50 mm KNO3. Thirty al of liposomes were added to 2ml of 100 mm citrate-phosphate buffer (pH 7.4) containing 20 i.M['4C]methylamine, and the same concentration of KCI or KNO3 as theliposomes, and either 0 (0) or 5 (@),Mm acridine orange. Accumulationof ['4C]methylamine was determined by filtering two 100 ,ul aliquots atthe times indicated. Results are the mean of two separate experiments.

either use other pH probes such as quinacrine or ['4C]methylamine,or ensure that acridine orange reports the same relative ApH asthese probes with a range of anions.The finding that mixtures of acridine orange and anions can

collapse ApH in tonoplast vesicles not only calls in to questionthe usefulness of this compound as a pH probe but also raisesdoubts about the physiological interpretations that have beengiven to effects of NO3- on pH gradients measured with acridineorange. The finding that the inhibition of the tonoplast ATPaseby NO3- can apparently be modulated by temperature (14) shouldnow be reexamined since we observed similar effects with thePPase but only when using acridine orange (Fig. 4). In addition,the suggestion that the tonoplast possesses a NO3-/H + symportthat exports NO3- from the vacuole must also be questionedbecause evidence for this system was obtained with acridine or-

ange. With this probe, Rea and Poole (26) showed that in redbeet tonoplast vesicles rates of PPi-dependent H + -pumping werelower with NO3- than with Cl-, even though neither anion di-rectly affected the PPase. Blumwald and Poole (3) suggested thatthis indicated the presence of a NO3 -/H + symport and conductedseveral experiments which seemed to support this. In particular,

they demonstrated that low concentrations of NO3-, but notCl-, added after the initiation of H+-pumping, initially stimu-lated but then dissipated pH gradients in beet tonoplast vesicles.The experiments of Rea and Poole (26) and Blumwald and Poole(3) were similar to those shown in Figures 1 and 6, respectively.In both cases, we found that results comparable to those origi-nally reported were only obtained with acridine orange andtherefore conclude that they are probably artefacts induced bythis pH probe. The possibility that acridine orange specificallyactivates the NO3-/H+ symport causing the observed increasein H+-leakage is excluded by the fact that similar effects werealso observed with liposomes which obviously do not possesssuch a transport system (Fig. 9).Although the experiments reported here raise doubts about

the existence of a NO3-/H+ symport at the tonoplast, Wang etal. (33), using quinacrine as the pH probe, found that snmallerPPi-dependent pH gradients were formed in oat root tonoplastvesicles in the presence of NO3-. If confirmed, this result mightindicate the presence of the symport. However, we and othershave been unable to repeat this observation (Fig. 1; 9, 25). Re-cently, Schumaker and Sze (28) have published results with[14C]methylamine as the pH probe which are consistent with thepresence of a general anion/H+ symport in oat root tonoplastvesicles. However, their results were also consistent with thepresence of an anion/H+ antiport which utilizes theApH to trans-port anions into the vacuole. Until these alternatives are clearlydistinguished we suggest that there is no firm evidence for theexistence of a specific NO3-/H+ symport at the tonoplast ofhigher plant cells.

In conclusion, results obtained using acridine orange as a pHprobe should be treated with caution as it probably induces leak-age of H+. Any results obtained with acridine orange shouldalways be checked using another pH probe. This is particularlyimportant in studies attempting to determine the existence ofH+-linked anion transport in plant membranes.

Acknowledgments-We are grateful to Drs. D. Sanders and P. A. Rea for usefuldiscussions.

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dissipation, gradients tonoplast vesicles and liposomes ... · forquin-acrine (final concentration...

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