Journal of Experimental Botany, Vol. 38, No. 189, pp. 642-648, April 1987

Vanadate Sensitive ATPase and Phosphatase Activityin Guard Cell Protoplasts of Commelina

M. D. FRICKER AND C. M. WILLMER

Department of Biological Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K.

Received 8 July 1986

A B S T R A C TFlicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPase and phosphatase activity inguard cell protoplasts of Commelina.—}. exp. Bot. 38: 642-648.

Phosphatase activity was measured in extracts of guard cell protoplasts of Commelina communis L.using the artificial substrate p-nitrophenylphosphate. A pH optimum of 5-8 to 6-3 was determined.Ammonium molybdate (Ol mol m"3) and sodium vanadate (1-0 mol m"3) gave almost completeinhibition of phosphatase activity at pH 60. ATPase assays were, therefore, conducted in the presenceof 0-2 mol m " 3 molybdate and vanadate was used as a specific inhibitor of plasmamembrane ATPaseactivity. Vanadate sensitive ATPase activity showed a pH optimum of 6-6 and activity was stimulatedby KC1. These properties are characteristic of plasmamembrane proton pumping ATPases in othersystems and suggest that proton extrusion in guard cells could be mediated by a similar enzyme. Themaximum ATPase activity is sufficient to account for all the proton flux observed during the stomatalopening response.

Key words—ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate.

Correspondence to: Department of Biological Sciences, University of Stirling, Stirling FK9 4LA,Scotland, U.K.

INTRODUCTIONThe opening response of stomatal guard cells is osmotically generated by the accumulationof potassium and concurrent uptake of chloride and/or synthesis of malate to maintainelectroneutrality within the cell (Zeiger, 1983). It has been proposed that these ion fluxes arechemi-osmotically coupled to a primary electrochemical gradient of protons which areextruded at the plasmamembrane (Zeiger, Bloom, and Hepler, 1978). In support of thishypothesis, proton efflux correlates well with stomatal opening and potassium accumulationin epidermal strips (Raschke and Humble, 1973; Gepstein, Jacobs, and Taiz, 1982/83), andhas also been implicated in blue light (Shimazaki, lino, and Zeiger, 1986) and CO2 responses(Gotow, Sakaki, Kondo, Kobayashi, and Syono, 1985) of guard cell protoplasts (GCP).Although the nature of the proton pumping system has not been determined yet, severalindirect lines of evidence suggest a plasmamembrane bound H+-ATPase is involved, withproperties similar to those characterized in other systems (reviewed by Sze, 1985; Marre andBallarin-Denti, 1985). Stomatal opening is inhibited by vanadate (Gepstein et al., 1982/83), aspecific inhibitor for plasmamembrane ATPases (Gallagher and Leonard, 1982), and stimu-lated by fusicoccin (Squire and Mansfield, 1974; Pemadasa, 1981; Clint and MacRobbie,1984). In addition fusicoccin also stimulates proton extrusion (regarded as a diagnosticfeature of H+-ATPase mediated processes—Marre, 1979; Rasi-Caldogno and Pugliarello,

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1985) in GCP (Shimazaki et al, 1986), with concomitant swelling (Schnabl, 1978; Clint,1985b) and potassium accumulation (Clint, 1985b). At the biochemical level, investigations ofATPase activity have been limited to extracts prepared from whole epidermal peels(Raghavendra, Rao, and Das, 1976), or plasmamembrane enriched fractions from wholeepidermis (Kasamo, 1979; Lurie and Hendrix, 1979). However, problems may arise whenattempting to relate results obtained with epidermal tissue to stomatal functioning in view ofthe unknown relative contributions from epidermal cells, subsidiary cells and guard cells(Outlaw, 1983). Since the development of methods for the routine isolation of GCP (Zeigerand Hepler, 1976; Fitzsimons and Weyers, 1983), biochemical investigations have beenpossible with highly purified cells. In this paper we are concerned with the detection ofa putative plasmamembrane ATPase in guard cell protoplasts isolated from Commelinacomtnunis L., using criteria established for H+-ATPases in other systems (Sze, 1985; Marreand Ballarin-Denti, 1985). Initial studies were directed at suppression of phosphataseinterference in ATPase assays through the use of selective inhibitors (Leigh and Walker,1980; Gallagher and Leonard, 1982).

MATERIALS AND METHODSPlant materialPlants of C. communis were grown from seed in John Innes No. 2 potting compost under greenhouseconditions for 6-8 weeks. Temperatures ranged from 13 °C (night) to 30 °C (day). Supplementarylighting (Thorn 125 W fluorescent tubes; Omega 150 W tungsten filament and Thorn, Kolorlux, 400 WMercury vapour lamps) provided a 16 h daylength and an energy fluence rate of about 110 /jmol m ~2

s"1 (400-700 nm) in the region of the plants.

Guard cell protoplast preparationGCP were isolated according to the method of Fitzsimons and Weyers (1983) with minor

modifications. Inclusion of 005% pectolyase (Birkenhead and Willmer, 1984) reduced protoplastrelease times to under 3 h and 20 mmol m " 3 CaCl2 was present in all media as recommended by Clint(1985a). To avoid membrane damage when harvesting the GCP from the digestion medium bycentrifugation, a Percoll cushion consisting of 90% Percoll, 300 mol m"3 mannitol and 10 mol m" 3

MES, pH 6-5, was used. To purify the protoplasts the Percoll gradient was reduced to two steps(90%/40%) and both layers contained 300 mol m"3 mannitol and 10 mol m"3 MES, pH 6-5. Releasedprotoplasts were transferred to 10cm3 300 mol m~3 mannitol containing 10 mol m"3 MES, pH 5-5 andwashed three times. The protoplast population was recorded in triplicate subsamples using animproved Neubauer haemocytometer and Leitz microscope (x 100).

Typically 1-56 GCP were prepared with viability greater than 95%, as judged by neutral red uptakeand general cell appearance.

Protoplasts were disrupted by resuspending the pellet in an ice-cold homogenization mediumcontaining 3-0 mol m"3 DTT, 3-0 mol m"3 EDTA, 0-1% w/v BSA, 0-5% PVP-40 and 50 mol m"3

MES, pH 6-5 and forcing the suspension through a 26 gauge needle. Light microscopy indicated allprotoplasts were broken by this procedure but chloroplasts remained intact. This sample was termedthe crude homogenate.

Enzyme assaysNon-specific phosphatase was assayed according to Leigh and Walker (1980) using hydrolysis of

p-nitrophenylphosphate (p-NPP) as substrate. The reaction medium (10 cm3) contained 50 mol m~3

MES, pH 60, 30 mol m~3 p-NPP and 10-20 mm3 sample, equivalent to about 104 protoplasts.Incubation was for 60 min at 30 °C (rates were linear for at least 90 min). To obtain pH curves thefollowing 50 mol m"3 buffers were used: citrate/KOH or acetate/KOH (between pH 3-5 and 50),MES/KOH (between pH 5-2 and 6-3) and HEPES/KOH (between pH 6-8 and 8-3).

ATPase activity was measured using the linked enzyme assay outlined by Auffret and Hanke (1981)at 30 °C in a Pye Unicam SP1800 dual beam spectrophotometer. The assay medium (1 0 cm3) containedthe following final concentrations; 30 mol m~3 MES/HEPES, pH 6-8, 3O mol m"3 MgCl2, 20 molm"3 PEP, 0-33 mol m~3 NADH, 3O mol m"3 DTT, 0Ol% BSA, 10 units pyruvate kinase, 14 unitslactate dehydrogenase (14 mm3 PK/LDH enzymes, Sigma), 0-2 mol m~3 ammonium molybdate and

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644 Fricker and Willmer—ATPase and Phosphatase in Guard Cell Protoplasts

50-100 mm3 crude GCP homogenate, equivalent to 5 x 10* protoplasts. The reaction was started bythe addition of ATP (final concentration 3-0 mol m~3). Vanadate sensitive activity was measured as thedifference in rates of N ADH oxidation in the presence and absence of 0-1 mol m " 3 vanadate. Potassiumstimulation was measured in the presence of 50 mol m~3 KC1. Activities were linear for at least120 min and allowed additions to be made to the cuvettes over this time period. To obtain pH curves,pH values were adjusted with Tris base or HC1 added to the cuvettes during the course of theexperiment. pH values were measured in situ with a Ml-410 Micro combination pH probe(Microelectrodes, Inc., U.S.A).

Ammonium molybdate and sodium vanadate were made up as 10 mol m " 3 stock solutions at pH 8-0and 10-5, respectively, to maintain the an ions in the monomeric state and aged for a minimum of 24 hto ensure complete dissociation of any polymers initially present (Goddard and Gonas, 1973; Kepert,1973).

Protein determinationThe soluble protein content of crude homogenates of GCP was measured using the method of

Bradford (1976).

R E S U L T S

In GCP extracts the pH optimum for non-specific phosphatase activity was between 5-8and 6-3 (Fig. 1). Maximum activities corresponded to 903±0-70 pmol h" 1 protoplast"1

(34 /xmol h " i mg" 1 protein). When acetate buffer was used below pH 5-5 the pH optimumwas broadened and extended to pH 5-5 (data not shown). In this respect it is recognized thatthe pH profile may be dependent on the type of buffer, the ionic strength and the ion speciespresent. However, we have not attempted further characterization as the major objective wasto inhibit non-specific phosphatase so that ATPase activity could be measured withoutinterference. For fixed pH studies MES/KOH buffer was used at a pH of 6-0. At this pH,

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3 . 5 4 .5 5 . 5 6.5 7.5 8.5

PHFIG. 1. pH profile of phosphatase activity in crude extracts of GCP; (•) extract without inhibitors;(A) extract plus 1 -0 mol m " 3 ammonium molybdate; (•) extract plus 1 -0 mol m "3 sodium vanadate. Each

point is associated with its standard error of the mean (s.e.); n = 4.

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Inhibitor Concentration

FIG. 2. Effect of varying inhibitor concentration on phosphatase activity in crude extracts of GCP(pH 6-0); (•) ammonium molybdate; (•) sodium vanadate. Each point is associated with its standard error

of the mean (s.e.) unless the s.e. is smaller than the point; n = 4.

molybdate gave 50% inhibition at 10 4 mol m 3 and almost complete inhibition at 10 1

mol m~3; vanadate had little effect below 10~2 mol m~3 but changed rapidly to givemaximal inhibition at 10 mol m~3 (Fig. 2). The extent of inhibition for both inhibitors (at10 mol m~3) was dependent on pH, with a marked decrease in effectiveness below pH 5-5particularly in the case of vanadate (Fig. 1). This loss of inhibitory activity was possibly due tocomplexation of vanadate by the citrate buffer as the effect was not observed with acetatebuffer (data not shown).

Vanadate sensitive ATPase activity both in the presence and absence of KC1 showed a pHoptimum around 6-6 (Fig. 3). Inclusion of 50 mol m"3 KG produced a pH dependentstimulation of activity, being maximal at lower pH values and decreasing as the pH wasraised (Fig. 3). Although observing the same pattern of activity in response to pH and KCL,the maximum potassium stimulated activity varied between experiments from 1-66 to2-52 pmol h"1 protoplast"1 (6-38 to 9-69 /amol h"1 mg"1 protein). However, 20% of thesevalues may be accounted for by the residual 5% non-specific phosphatase activity remainingeven in the presence of molybdate (Figs 1, 2).

DISCUSSIONHydrolysis of the artificial substrate p-NPP is generally accepted as indicative ofphosphatase activity of low substrate specificity. Two groups of phosphatases showingactivity towards a broad spectrum of phosphate esters have been defined by their pH optima:acid phosphatase (E.C. 3.1.3.2) having optima between 50 and 6-5 and alkaline phosphatase(E.C. 3.1.3.1) having optima between pH 7-6 and 9-9 (Sexton and Hall, 1978). In GCP the

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64© Flicker and Willmer—AT Pose and Phosphatase in Guard Cell Protoplasts

value obtained was towards the high end of the acid phosphatase range (Fig. 1), butbroadened to more acid values if acetate rather than citrate buffer was used (data not shown).Similar effects of buffers have been reported in animal systems (Hollander, 1971). Alkalinephosphatase activity was insignificant in GCP, in common with other reports on its activityin other cell or tissue types (Sexton and Hall, 1978).

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FIG. 3. pH profile of vanadate sensitive ATPase activity in crude extracts of GCP in the absence (•), orpresence (•), of 50 mol m " 3 KC1. Activities are expressed as a percentage of the maximum activity in the

absence of K.O. Each point is associated with its standard error of the mean (s.e.); n = 5.

Schnabl and Kottmeier (1984) localized acid phosphatase exclusively in the vacuoles ofViciafaba L. GCP using cell fractionation techniques, though levels were 7-8-fold lower ona protoplast basis than those observed in this study with C. communis.

The inhibitor studies indicate molybdate was an effective inhibitor of phosphatase activityat low concentrations as reported by other authors (Leigh and Walker, 1980), whilstvanadate was only inhibitory at higher concentrations (Fig. 2). This pattern of differentialinhibition has been observed in other systems (Gallagher and Leonard, 1982). In additionthere was a substantial level of phosphatase activity below pH 5-5 which was insensitive toboth molybdate and vanadate and whose origin is uncertain. Above pH 5-8 inclusion of0-2 mol m" 3 molybdate was sufficient to inhibit phosphatase activity by more than 95%(Figs 1, 2) and allowed vanadate to be used as a selective inhibitor of the plasmamembraneH + -ATPase with little interference from phosphatase activity.

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Fricker and Willmer—ATPase and Phosphatase in Guard Cell Protoplasts 647

The pH optimum and pH profile for potassium stimulation of the vanadate sensitiveATPase activity in homogenates of GCP were similar to plasmamembrane ATPases presentin corn leaf mesophyll (Perlin and Spanswick, 1980), corn roots (Gallagher and Leonard,1982), red beet (Briskin and Poole, 1983) and tobacco epidermis (Lurie and Hendrix, 1979).The maximum K+-stimulated ATPase activity obtained in our study (2-52 pmol proto-plast" 1h"1) equates with a proton flux of 750 or 1 500 nmol m"2 plasmamembrane s"1,depending on whether a stoichiometry of 1 or 2 is used for the number of protons pumpedper ATP hydrolysed. Information on the H+/ATP ratio in other systems suggests a value oftwo may be energetically feasible, but a stoichiometry of one may allow kinetic controlindependently of the energy supply (Smith and Raven, 1979; Spanswick, 1981). These figuressuggest that the proton pumping capability of the ATPase is more than adequate to accountfor the observed K+ fluxes in GCP from C. communis (24-115 nmol m~2 s"1, Fitzsimonsand Weyers, 1986), or rubidium fluxes in 'isolated' guard cells (10-30 nmol m~2 s~\MacRobbie, 1981). Unequivocal demonstration of a plasmamembrane H+-ATPase requiresisolation and purification of a plasmamembrane fraction with proton translocatingcapability. Using vanadate sensitive ATPase activity as a marker, we are now involved inseparation of plasmamembrane fractions for further characterization.

A C K N O W L E D G E M E N TWe thank the Science and Engineering Research Council for financial support for M.D.F.during the course of this work.

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FITZSIMONS, P. J., and WEYERS, J. D. B., 1983. Separation and purification of protoplast types fromCommelina communis L. leaf epidermis. Ibid. 34, 55-66.

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GALLAGHER, S. R., and LEONARD, R. T., 1982. Effect of vanadate, molybdate and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots. Plant Physiology, 70,1335-40.

GEPSTEIN, S., JACOBS, M., and TAIZ, L., 1982/83. Inhibition of stomatal opening in Viciafaba epidermaltissue by vanadate and abscisic acid. Plant Science Letters, 28, 63-72.

GODDARD, J. B., and GONAS, A. M., 1973. Kinetics of the dissociation of decavanadate ion in basicsolutions. Inorganic Chemistry, 12, 574-9.

GOTOW, K , SAKAKI, T., KONDO, N., KOBAYASHI, K., and SYONO, K., 1985. Light-induced alkaliniza-tion of the suspending medium of guard cell protoplasts from Viciafaba L. Plant Physiology, 79,825-8.

HOLLANDER, V. P., 1971. Acid phosphatases. In The Enzymes. Ed. P. D. Boyer. Academic Press,London and New York. Pp. 449-98.

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KASAMO, K., 1979. Characterization of membrane-bound Mg++-activated ATPase isolated from thelower epidermis of tobacco leaves. Plant and Cell Physiology, 20, 281-92.

KEPERT, D. L., 1973. Isopolyanions and heteropolyanions. In Comprehensive inorganic chemistry.Volume IV. Eds J. C. Bailar, Jr., H. J. Emeleus, R. Nyholm and A. F. Trotman-Dickenson.Pergamon Press, Oxford. Pp. 607-72.

LEIGH, R. A., and WALKER, R. R., 1980. ATPase and acid phosphatase activities associated withvacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta, 150, 222-9.

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MACROBBIE, E. A. C , 1981. Ion fluxes in 'isolated' guard cells of Commelina communis L. Journal ofExperimental Botany, 32, 545-62.

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OUTLAW, W. H., 1983. Current concepts on the role of potassium in stomatal movements. Physiologiaplantarum, 59, 302-11.

PEMADASA, M. A., 1981. Abaxial and adaxial stomatal behaviour and responses to fusicoccin onisolated epidermis of Commelina communis L. New Phytologist, 89, 373-84.

PERUN, D. S., and SPANSWICK, R. M., 1980. Labelling and isolation of plasma membranes from cornleaf protoplasts. Plant Physiology, 65, 1053-7.

RAGHAVENDRA, A. S., RAO, I. M., and DAS, V. S. R., 1976. Adenosine triphosphatase in epidermaltissue of Commelina benghalensis: possible involvement of isozymes in stomatal movement. PlantScience Letters, 7, 391-6.

RASCHKE, K., and HUMBLE, G. D., 1973. No uptake of anions required by opening stomata of Viciafaba: guard cells release hydrogen ions. Planta, 115, 47-57.

RASI-CALDOGNO, F., and PUGUARELLO, M. C , 1985. Fusicoccin stimulates the H + -ATPase ofplasmalemma in isolated membrane vesicles from radish. Biochemical and Biophysical ResearchCommunications, 133, 280-5.

SCHNABL, H., 1978. The effect of Q " upon the sensitivity of starch-containing and starch-deficientstomata and guard cell protoplasts towards potassium ions, fusicoccin and abscisic acid. Planta,144, 95-100.and KOTTMEIER, C , 1984. Determination of malate levels during the swelling of vacuoles isolatedfrom guard cell protoplasts. Ibid. 161, 27-31.

SEXTON, R., and HALL, J. L., 1978. Enzyme cytochemistry. In Electron microscopy and cytochemistry ofplant cells. Ed. J. L. Hall. Elsevier/North Holland Biomedical Press. Pp. 63-147.

SMMAZAKI, K., IINO, M., and ZEIGER, E., 1986. Blue light dependent proton extrusion by guard cellprotoplasts of Vicia faba. Nature, 319, 324-6.

SMITH, F. A., and RAVEN, J. A., 1979. Intracellular pH and its regulation. Annual Review of PlantPhysiology, 30, 289-311.

SPANSWICK, R. M., 1981. Electrogenic ion pumps. Ibid. 32, 267-89.SQUIRE, G. R., and MANSFIELD, T. A., 1974. The action of fusicoccin on stomatal guard cells and

subsidiary cells. New Phytologist, 73, 433-40.SZE, H., 1985. H+-translocating ATPases: advances using membrane vesicles. Annual Review of Plant

Physiology, 36, 175-208.ZEIGER, E., 1983. The biology of stomatal guard cells. Ibid. 34, 441-75.

BLOOM, A. J., and HEPLER, P. K., 1978. Ion transport in stomatal guard cells: a chemi-osmotichypothesis. What's New in Plant Physiology, 9, 29-32.and HEPLER, P. K., 1976. Production of guard cell protoplasts from onion and tobacco. PlantPhysiology, 58, 492-8.

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