densa ATP-Dependent

Ethanol-induced Activation of ATP-Dependent Proton

Extrusion in Elodea densa Leaves'

Maria T. Marre*, Alberto Venegoni, and Anna Moroni

Centro di Studio

del

Consiglio

Nazionale delle Ricerche sulla

Biologia

Cellulare eMolecolare delle

Piante

c/o Dipartimento di Biologia, Universita degli Studi

di

Milano,

Via Celoria

26,

20133

Milano,

Italy

ABSTRACT

In Elodea densa leaves, ethanol up to 0.17 M stimulates H extrusion activity.This effectisstrictly dependentonthe presence of K' in the medium and issuppressed by the presence of the plasmalemma H"-ATPase inhibitor vanadate. Stimulation of H+

extrusionisassociated with(a)adecreaseincellularATPlevel, (b) amarkedhyperpolarization of transmembrane electricalpotential, and (c) an increase in netK' influx. These results suggest that ethanol-induced HW extrusion is mediatedbyan activationof the plasma membraneATP-dependent, electrogenicprotonpump.This stimulating effectisassociated withanincrease ofcellsappHand ofthe capacitytotakeup the weakacid5,5-dimethyloxazolidine- 2,4-dione, whichisinterpretableasdueto anincrease ofcytosolic pH.Thisindicates that the stimulationof H' extrusion by ethanol doesnotdependon acytosolic acidificationby productsof ethanol metabolism. Thesimilarityofthe effectsof ethanol andthose of photosynthesisonproton pumpactivityin E.densaleaves suggests that a commonmetabolicsituation isresponsiblefor the activation oftheATP-dependentHW-extrudingmechanism.

ATP-dependentH+ extrusionisregulated in vivoby var-

iousfactors, including cytosolic pH (8, 21), Em2 (16, 22), and naturalendogenous andexogenousfactors suchashormones andphytotoxins (13). More recentlythe conclusion that the stimulating effect of lighton H+ extrusion in Elodea leaves might be mediated by photosynthesis-induced metabolic changes (14) has emphasized the need to understand the relationship between metabolism and activity of the H+

pump.

Insearch of factors that may influence the proton pump

of Elodea densa leaves, ^we have focused our attention on

certainglycolytic metabolites includingsugars,organic adds, glycerol, and ethanol. Among these, ethanol was found to inducein E. densa leavesamarked, reproduciblestimulation of thecapacitytoacidifythe medium.

Astimulating effect of ethanol onthereduction ofextra- cellularelectronacceptorsandontheH+extrusion associated withithasbeenreported byCraig and Crane (6), Chalmers et al. (4), and Bottger and Luthen (3), and it has been

'Researchsupportedbythe NationalResearch Council ofItaly, SpecialProject RAISA, SubprojectNo.2,PaperNo.383.

2Abbreviations:Em, transmembraneelectricalpotential;FC,fusi- coccin; BTP, 1,3-bis(tris(hydroxymethyl)methylamino)-propane;

DMO,5,5-dimethyloxazolidine-2,4-dione; K+0,extracellularK+.

interpretedas being due to thealcohol dehydrogenase-me- diated rise of NADH, as an electron donor to the plasma membraneredox system (10). Astimulation ofH+extrusion byethanol andproponolintheabsence ofartificial extracel- lular electron acceptors has been reported by Bottger and Luthen(3).

Moving from these results, the main objectives of the presentinvestigationswere(a) toobtainageneraldescription of the effect of ethanolon H+ extrusion in E. densa leaves, (b) to ascertain whether it is mediated by the ATP-driven protonpump, and(c) to acquire somepreliminaryinforma- tionconcerning itsmechanism: inparticular, whetheror not thestimulation of H+ extrusionby ethanol might be due to cytosol acidificationand/orEmdepolarization(as seen in the caseofferricyanide reduction by the plasma membrane redox system) (12, 19, 28).

MATERIALS AND METHODS

Elodea densa plants were cultivated in large tanks in a greenhouse (19). Actively growingshoots(about 5 cm long) were collectedin the morning and kept under runningtap waterforabout1 h. Theleaves were then excised, random- ized, and pretreated for2 hin0.5 mm CaSO4 inthe darkat 200CinagitatedErlenmeyerflasks(18leaves=150 mgfresh weightin 10 mL) andthen treated asdescribedindetail for each experiment. Whenrequired,FC,Na-orthovanadate,and DCMUwereaddedtothe CaSO4solution 30min beforethe experiment.

pH andH'ExtrusionMeasurements

After pretreatment, samples (150 mgfresh weight/7mL) were transferred to a solution containing 0.5

mt

Mes (pH 5.5withBTP),0.5 mmCaSO4,1 mmK2SO4. Variations inthe testedsolutions are indicatedinfigure legends. The experi- mentswereconductedintriplicateat200C eitherinthe dark (inthe presence of5

/M

DCMU)or in thelight (50

W/m2).

Proton extrusion wasmeasured atdifferent times by back- titrationofthe external mediumafterremovalof

CO2

accord- ing to Lado et al. (11). The titration curves (between pH 4 and 7) were also used to measureeventual changes of the

buffering

capacityofthe medium due to thereleaseof weak acids fromtheleaves.

K+Uptake Measurements

Changes in K+ concentration in the medium were deter- mined by atomic absorption

spectrophotometry (Varian

1120

Techtron AA 1275).Basal medium contained10 mm Mes (pH 5.5 with BTP), 0.5mm CaSO4, 0.25 mm K2SO4, 5

lAM

DCMU with and without 0.2mm Na-orthovanadate.

Em Difference

MeasurementsofEmwereperformed by the microelectrode procedure described byMarre etal. (17). Thebasal medium contained 1 mm Mes (pH 5.5 with BTP), 0.5

mt

CaSO4, 1 mM K2SO4, 5

uM

DCMU. Variations in the medium compo- sition aredescribedinthelegendstothe figures.

Cell

Sap pH

When treatments were terminated, leaves were washed in distilledwater,blottedonfilterpaper,transferredintoplastic syringes, andfrozenat

-300C

foratleast3 h.After

freezing

andthawing, the cellsap(about0.2mL)was

pressed

outof the syringeand thepH was measuredbymeansofa Radi- ometerpH meter

equipped

with a flat tip electrode

(Ingold

40424, lot 40330-M8).

Accumulation of

DMO

and Evaluation of Cytoplasmic

pH The reliability of the weak acid-weak base distribution method for the evaluation of cytosolic and vacuolar pH in Elodea leaves has been demonstrated by

Beffagna

and Ro- mani (2). In our experiments, the weakacid usedas a probe was

[2-_4C]DMO

(155

kBq/,umol).

For DMO accumulation afterpretreatments, thesamples (150 mgfresh weight)were transferredto10 mLofasolutioncontaining10 mm Mes(pH 5.5with BTP), 0.5 mmCaSO4, 5

AM

DCMU,5

AM

DMO,

1%

(v/v)ethanol (in the presence or absenceof1 mmK2SO4).At theendof the incubation with DMO, the leaveswererinsed with the

corresponding

solution without label for 3 min at

0°C,

thenwashed with water, blotted

dry,

and

placed

into the scintillation vialsfortissuedigestion and

bleaching

prior todeterminationof their

radioactivity.

The pH values of 'bulk cytoplasm' were calculated from the accumulation of DMO by the weak acid distribution method as described by Beffagna and Romani(2). The pH values hadbeencalculatedutilizing the previously measured cellsap pH value asvacuolar pH and assuming that DMO distribution had reached

equilibrium

after 90 min of treatment.

Determination

of Metabolites

After pretreatment, samples (150 mg fresh weight) were transferredto7 mLofasolution containing10 mm

Mes/BTP

buffer(pH 5.5), 0.5 mmCaSO4,5 MmDCMU,0.1 mm

FC,

1%

ethanol (with orwithout 1 mmK2SO4). At theend oftreat- ments, leaves were washedindistilled water, blottedonfilter paper, and frozeninliquid nitrogen. Malatewasdetermined enzymically by the malate

dehydrogenase/citrate

synthase method as described by Stitt et al. (29). The intracellular content of ATP was measured

by

the

luciferin/luciferase

method asdescribed byNovackyetal. (24).

All the experiments wererun intriplicate and repeatedat least three times. The data presented are those ofa typical experiment in which the coefficient of variation did not exceed± 6%.

RESULTS

Effects of Some Metabolites and Alcohols on External pH In a preliminary series of experiments, we measured the effects of some metabolites and alcohols on H+ extrusion, measured by titration after 60minof incubation in a medium containing 1 mM K2SO4, 0.5 mm Mes-BTP, pH 5.5, 0.5 mm CaSO4, 150 mgfresh weightofleaves in 7 mL. Among the compounds tested, 0.17M ethanol and propanol induced a well-marked stimulation of H+ extrusion, whereas at the same concentration, methanol, buthanol, and isopropanol had no significant effect, andpentanol induced an alkalinization of the medium.Fiftymillimolar glucose and 0.2Mglycerol were ineffective(data not shown).

Characterization of the Effects of Ethanol on H+ Extrusion As shown in Figure 1, ethanol, in the presence of 1mm

K+,

markedly stimulatedH+ extrusion. The maximumeffectwas observed for the 0.17 M concentration, already significant after 15 min of treatment, and increased roughly linearly with time. The titration curves ofthe incubation media be- tween pH 4 and 7 indicated that ethanol, up to 0.5 M, did notinduce any significant differencein the buffer capacity of the medium (e.g. by acetic acid arising from ethanol oxidation via alcohol dehydrogenase). This ruled out the possibility ofanalcohol-induced release oforganic acids by the leaves.Atethanolconcentrationshigherthan 0.17

M,

the stimulating effect decreasedtobecome strongly inhibitoryat the 1.7 M concentration. The absence of

K+0

completely suppressed H+ extrusion both in the controls and in the ethanol-treated samples (Fig. 2). Because ATP-dependent proton extrusion requires the presence of K+ (or of other permeant,depolarizing cations) inthe medium(15, 16, 22), thisfinding suggestedaninvolvementof the ATP-dependent

6 - Et 0.17 M

5 ^- sEt0.51M

4 - o / Et 0.08M

3

LL 2 Control

E -1-

E

+ -2

Et 1.7M

I -4_

15 30 60

TIME (min)

Figure 1. Effect of increasing concentrations of ethanol (Et) on apparentH+ extrusion(-AH+). Control:0.5mmMes/BTP, pH 5.50, 0.5mMCaSO4,1 mMK2SO4,5,M DCMU. Barsindicate± SE.

2

4 6 8 10

12

Figure2. Effect ofK+ and vanadateonethanol-induced H+extru- sion. Control: 0.5 mm Mes/BTP, pH 5.5, 0.5 mm CaSO4, 5 ,uM DCMU.When present: 1 mmK2SO4, 0.2 mmvanadate (V),0.17M

ethanol(Et). Barsindicate^±SE.

H+-ATPase. Thishypothesis is supported byresultsreported in Figure 2, showing that 0.2 mm vanadate, a relatively specific inhibitor of the plasmalemma H+-ATPase (see ref.

1), completely suppressed both the basal and the ethanol- stimulatedH+ extrusion.

Interactions between Ethanol and FCand betweenEthanol andLight in their EffectonH' ExtrusioninElodea Leaves

The analysis of the interactions among different factors influencingasingleprocessmight be of help inunderstand- ing their mechanism of action.Afurther seriesofexperiments

wasthus aimedatdefining the interactions between ethanol and other factors knowntoinfluenceH+extrusionin Elodea leaves, suchasFC andlight.

Previous results had shown that FC and light are some-

what, although not completely, additive in promoting H+

extrusion (14). The data reported in Figure 3 confirm this result and also show that^someadditivityisobserved between the effects of FC andethanol, whereasnoadditivityatall is

seen between those of ethanol and light. In fact, in our

conditions thelight-induced stimulationofK+-dependent H+

extrusion was not further enhanced by the presence of ethanol.

Changes of Emand ofK' Uptake Associated with Ethanol- Induced H' Extrusion

As shown in Figure 4, the addition of 0.17 M ethanol induced a significant hyperpolarization (35-45 mV) of Em, already detectable after a few minutes of treatment and reaching its maximum after about 15 min. This ethanol- inducedhyperpolarizationwascompletelyreversed by wash- ing andwassuppressed by the addition of the protonophore uncoupler carbonylcyanide p-trifluoromethoxyphenylhydra-

zoneandby thepresenceof theH+-ATPase inhibitor,vana-

-A

H

+

(pmol /

^g

FW * h )

Figure3. Effect of FC and lightonbasal- and ethanol-induced H+

extrusion.Basal medium(control):0.5mmMes/BTPpH 5.5, 0.5mm CaSO4. When present: 1 mmK2SO4, 0.17 M ethanol, 0.1 mm FC.

dark, 5AMDCMU inthemedium; light, 50W/m2. Barsindicate+ SE.

4min

WASH ING ETHANOL

170mM +

-150mV _ -153mV

I5OmV

ETHANOL -230mV

170mM

c FCCP

C-K ~~~5 10-6M

-200 mV

ETHANOL

170mM -185mV

<)+K+ /

~-145mV VANADATE

2*10-4M ETHANOL

+K4 l 170mM

-16m

-150mV

Figure 4. Ethanol-induced changes in membrane potential. Two leavesweremaintainedin anaerated solution, thermoregulatedat

20°C (continuous flow 15mL/min). C-K+, 1 mmMes/BTP, pH 5.5, 0.5 mM CaSO4,5,M DCMU;+K+,+ 1 mmK2SO4. Whenpresent:

0.2 mm vanadate (V), 5 gm FCCP (carbonyl cyanide p-trifluoro- methoxyphenylhydrazone). Ethanol was added at 0.17 M final concentration.

E

4.

1

2

1

0

-1 -2 -3

///.100.1' Dark Z

10C

^ontrol

I e , //+

Light

a

I Control + Light

.-

I

^{Light +Fusicoccin}

Ethanol+ Light

II I I I I~~~~~~~~~~

i

~12

L.L

0

4:-

0-

C V Et Et+V

Figure 5. Ethanol-inducedchangesinnetK+influx.Controlsolution (C): 10mm Mes/BTP, pH 5.5, 0.5 mmCaSO4, 5 ,uM DCMU, 0.25 mM K2SO4. When present, 0.2 mmvanadate (V). Ethanol(Et) was added at 0.17 M final concentration. Barsrepresent±SE.

date.This ethanol-induced hyperpolarization occurred both in the absence and in the presence of K+ in the external medium (Fig. 4, a and b), a behavior repeating the one previously reported fortheeffects of FC and light(14).Thus, the presence of K+ in the medium is also

required

for the effectof ethanolonH+ extrusionbutnotforthat onEm. The simplest interpretation (in this case as in those of FC and lightactivationof H+ extrusion)isthatinthe absenceof

K+o,

ethanol is still able to stimulate the H+-ATPase, but the hyperpolarization (consequenttotheextrusionofanamount of H+toosmalltobe

measured) rapidly

inhibits the

H+

pump (22).

ThedatareportedinFigure5show thatthehyperpolariz- ingeffect of0.17Methanolwasassociated witha

significant

x

(A'3,

6.35 6.30 ^- 6.25 ^- 6.20 ^- 6.15 ^- 6.10 6.05.4

6.00 ^- 5.95 - 0

increaseofK+uptake, thus repeating, once again, a feature already observedinthecasesof theactivationofH+extrusion by FC and by light (13, 14). Vanadatecompletelysuppressed thestimulation ofK+uptakebyethanol.

Intracellular

pHChanges

Induced by Ethanol

The

possibility

that ethanol might have influenced H+

extrusion byacidifyingthecytoplasmwassuggestedby pre- vious reports showing that H+ transport is stimulated, both invivoandinvitro, byadecreaseof cytosolicpH inthepH 8 to 6.5range(23, 26, 27). Thepossibilityseems toberuled outby theresultsoftheexperiments reportedin Figure 6A, showingthat 0.17Methanol,inthepresenceof1 mmK2SO4, induced a progressive alkalinization of the cell sap already

significant

after15minoftreatment, whereas a muchsmaller increasefromtheinitial pH wasinducedby K+ alone. In the absenceof

K+0,

aslightacidificationwasobserved, independ- entof thepresence orabsenceof ethanol.

Cellsap pH can beconsideredameasurementof vacuolar pH (16). A more specific, although essentially qualitative, evaluation oftheinducedchanges in

'bulk cytoplasmic'

pH was obtained by the weak acid distribution method. As shown in Figure 6B, the simultaneous presence of ethanol andK+ significantly enhanced the rateofDMO uptakeand inducedamarkedincrease in the timerequired for the out- in equilibration of the probe, thus indicating a progressive alkalinizationof the cytosol. Intheabsenceof

K%,

nosignif- icant differencein DMO accumulation was found between the ethanol-treated andtheuntreatedsamples. A calculation of cytosolic pH (see

'Materials

and

Methods')

showed that ethanol and K+ are synergic in their alkalinizing action, as was also observed for the H+ extrusion-stimulating action (seeFig. 2).

Changes

in

Intracellular

ATP

and Malate Levels

The data of Table I show that 0.17 M ethanol, in the presenceofK+,markedly depressedthe ATP level measured

U.

--

0)

-

0

E

c

0 0 E

co

0

10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90

TIME(min)

TIME (min)

Figure6. Ethanol-induced changes in cell sap pH (A)and DMOaccumulation (B) in the absenceand in the presence of 2 mmK+ inthe medium. ThecytoplasmicpHvalues,calculatedat 90min,arereportedintheinset.Control medium(C), 10mmMes/BTP, pH 5.50,0.5mM CaSO4,5,mDCMU; K+, control+ 1 mmK2SO4. When present,0.17M ethanol(Et). DMOwasaddedat5lM(155kBq/fumol)concentration.

Barsrepresent±SE.

Table I. Effect of EthanolonIntracellularATPand Malate Levels Control: 10mmMes/BTP pH 5.50, 0.5 mmCaSO4,5jAm DCMU, 1 mM K2SO4. When present, 0.17 Methanol and 10-4 M FC. Data aremeansof three replication±SE

ATP Contentat MalateContent 60 min at60min

nmol/gfreshwt Amol/gfreshwt

Control(-K+) 140± 5 7.76±0.11

+ K2SO4 135± 1 8.5±0.3

+Ethanol 158± 7 8.61 ± 0.31

+Ethanol + K+ 110±4 10.5 ±0.3

+FC 150±5

+ FC +K+ 108±1

inthe leaves after 60 min oftreatment. This effectwasvery

similarto that induced by FC + K+, in full agreement with the results previously reported by other authors and inter- preted asbeing due to the increased utilization of ATP by the H+-extruding ATPase (31). The slight increase of ATP inducedby ethanol in the absence of

K+.

(no H+ extrusion occurring) might be duetotheeasyutilization of ethanolas a respiratory substrate by Elodea leaves (V. Trockner, M.

Marre,

unpublished data).

Anincrease in malatelevel, associated with the stimulation of H+ extrusion by treatments with FC, IAA, or light, has beenreported in various materials, and ithasbeeninterpreted

as depending on the stimulation of phosphoenolpyruvate carboxylation (9, 20, 30). The data of TableIshow that also inthecaseoftreatmentwith 0.17Methanol, the stimulation of H+ extrusion is associated with an increase by 25% of intracellular malate. It is worthy to note that, given the distribution (mainly vacuolar) to malate in the cell, this moderate increase of 'bulk'malate concentrationmightcor-

respondtoamuchlargerpercentageincreaseof this metab- oliteinthecytosol (25).

DISCUSSION AND CONCLUSION

The results of the present investigation show that in E.

densaleaves, ethanol markedly stimulatesapparentH+extru- sion, with a maximum effect at the 0.17 M concentration, higher concentrations becoming progressively inhibitory.

Thiseffect^seemsrelatively specificfor ethanol andpropanol.

Other alcohols tested^areeither inactive (methanol, butanol, isopropanol)orinhibitory (pentanol). Among glycolyticme-

tabolitestested, glucoseisinactive,whereasglycerolinduces

someslight stimulation of H+ extrusion.

In our experimental conditions, the effect of ethanol on

apparentH+extrusionwasstrictly dependentonthepresence

ofK+0 andwasassociated with(a)anincrease inK+ uptake;

(b)ahyperpolarizationofEm; (c)analkalinization of the cell

sapand of the cytosol; (d)an increase in malate level; and (e) a decrease in ATP level. Previous work with FC had shown thatallof theseresponsescanbeinterpretedasbeing due to a stimulation of the plasmalemma H+-ATPase (13).

Theinterpretation that ethanol-inducedH+extrusion isme-

diatedbyan activation of the ATP-driven H+pumpis also supported by its inhibition by the H+-ATPase inhibitor, vanadate.

The magnitude ofthe effect of ethanol in promoting H+

extrusionand associated responses in Elodea leaves is similar tothose of eitherFCorlight.Allavailable evidence pointsto theconclusion that the enhancement ofH+ extrusionby FC, light,orethanolismediatedby an increase in activity of the plasmalemmaH+-ATPaseand that it is not a consequence of eitheradepolarizationofEm or anacidification ofthecytosol.

Amongtheexperimentsreportedinthispaper,thoseinwhich thesethree stimulating factors have beencombined together haveshowna partial additivity for ethanol andFC, aswell asforlight and FC,suggestingthat the mode ofactionof FC is insome waydifferent from that of either ethanolorlight.

On the contrary, no additivityatall wasobserved between light and ethanol, suggesting a strictsimilarityin the mech- anismsofactionof thesetwofactors.

In the case of both factors, the simplestinterpretationof theactivationof thepump seems tobethatit ismediated by some change in metabolism able to influence the state of activationof theH+-ATPase intheplasmamembrane. In the caseof photosynthesis-inducedactivation, weconsidered the possibility ofarelationship between thiseffect and the light- inducedincreaseof malate level(7). It is interesting that an increase in malate (presumably via acetylCoA [5]) is also observed when H+ extrusion in Elodea leaves is stimulated by ethanol. Changes in malate level or, more generally, in the metabolicarea towhich malatebelongs, arepresumably associated withchangesinimportantregulatoryfactors,such as,for example, pyridincoenzymes and thiol compounds (for discussionseerefs. 17and 20). Further researchisobviously requiredtothrowlightonthis and otherpossibilities.

ACKNOWLEDGMENTS

We aregrateful to Professor ErasmoMarrefor useful advice and critical reading of the manuscript. We also wish to thank Dott.

NicolettaBeffagna for help with cytoplasmic pH calculations.

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