Plant Cell Reports

Plant Cell Reports (1985) 4:311-314

© Springer-Verlag 1985

Cation stimulation of the proton-translocating redox activity at the plasmalemma of C a t h a r a n t h u s roseus cells G~rard Marigo and Mouhssine Belkoura Centre de Physiologie V6g6tale de l'Universit6 Paul Sabatier, Laboratoire Associ6 au C.N.R.S. n ° 241, 118 Route de Narbonne, F-31062 Toulouse Cedex, France Received June 13, 1985 / Revised version received October 7, 1985 - Communicated by P. Matile

ABSTRACT Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact eel _ s and isolated Ca2~ protop!asts, by the ability of and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP , SDS-) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH. ABBREVIATIONS + CP : eetylpyridinium ; EGTA : ethylene glycol bis (~-aminoethyl)-N,N'-tet~aacetic acid ; FeCN : potassium ferricyanide ; SDS : sodium dodecyl sulfate ; SHAM : salicylhydroxamic acid INTRODUCTION In plant cells, it is generally accepted that an electrochemical proton gradient can be established by a plasmalemma ATPase (Spanswick, 1981). Recent reports have described a transplasmalemma redox system in various eukaryotic cells (Chalmers and Coleman, 1983 ; Federieo and Giartosio, 1983 ; Craig et al. , 1984 ; Lin, 1984 ; Rubinstein et al. , 1984 ; Sijmons et al. , 1984 ; Barr et al. , 1985) and in a few cases evidence has been presented for a direct link between this redox system and the generation o f an electrochemical proton gradient (Lin, 1984 ; Sijmons et al. , 1984). If proton fluxes across the plasma membrane are at least partially mediated by a redox activity, this will have important implications on determining the regulation of solute and ion transport. A proton-linked redox system might also play a role in the maintenance of the extracytoplasmic pH, the control of which is of great importance in natural processes such as growth and also the maintenance of the extraeellular pH which may have direct implications on the biotechnological utilization of

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plant cell cultures. We investigated the possibility of extracellular pH control by a proton redox pump using C. roseus cells. Our results indicate that a redox system localized at the plasmalemma can drive a proton efflux, and that it is stimulated by different cations. ~TERIALS

AND METHODS

The experiments were performed with cell suspension cultures of the cell line C 20 of Catharanthus roseus (L.) C. Don. The characteristics of the culture conditions have been previously described (Marigo et al. , 1983). Cell protoplasts were isolated from 7-day-old cells as already described (Marigo et al. , 1985) and suspended in a simple 0.7 M mannitol medium. Transmembrane ferricyanide reduction was followed : (I) in cell suspension cultures by spectrophotometric measurements at 420 run of reduced ferricyanide. Assays were performed in 50 ml Erlenmeyer flasks placed on a shaker. The incubation medium containing the cells (about 3 g fresh wt. in a final volume of 20 ml) was 50 mM Tris-Mes pH 7.5, with 5 mM ferricyanide. Aliquots of 2 ml were removed at specified times and immediately stored in a. bath at 0°C before centrifugation at the same temperature to spin down the cells. Determinations were performed on the supernatant after dilution by a factor 5. (2) on a protoplast suspensions by a spectrophotometric assay. Measurements were performed with a Kontron Uvikon model spectrophotometer ; the reaction was followed by the decrease in absorbanc~ at 420 nm. A typical reaction mixture contained i0 protoplasts, in a final volume of 3 ml, suspended in 5 % Ficoll to avoid their sedimentation, 1 mM ferricyanide, 0.7 M mannitol, 25 mM Tris-Mes pH 7.5 and variable quantities of salts. A millimolar extinction coefficient of 1 was used for calculating the rate of fer$icyanide reduction. The H efflux was evaluated directly by the pH changes of the incubation medium not buffered for this experiment. A f t e r 20 min preincubation, to stabilize the pH of the medium, the reaction was started by the addition of ferricyanide, pH Changes were measured at specific times with 20 ml of a cell suspension containing about 3 g of cells. In the case of protoplast suspensions, for which the pH changes are more rapid, the pH values of the medium were monitored continuously. Protoplasts were maintained in suspension in a vial placed on a

312 rotary shaker ; the reaction vial contained 0.I ml of protoplast suspension (about 3.100 protoplasts) and 0.9 ml of 0.7 M mannitol. Measurements were performed with a Beckman model pH I 70 pH meter, connected to a potentiometric recorder. The recorder deflection was calibrated by addition of i00 nmol of HCI to each protoplast suspension. For the preparation of a microsomal fraction, intact protoplasts were broken by osmotic swelling (by resuspending in a medium containing 0.4 M sorbitol, 0.2 % albumine bovine, Hepes buffer pH 8) and successive passes of the suspension through a hypodermic needle. The microsomal fraction was then isolated by sedimentation between 12,000 g (20 mins) and i00,000 g (60 min ).

pH 5,6

5.0

4.4

I 15

RESULTS AND DISCUSSION Evidence for a proton translocating redox activity at the plasmalemma of C. roseus cells Cells of C. roseus incubated with ferricyanide cause its reduction correlatively with an increased proton extrusion from the cells (Fig. i). These processes are probably mediated by an enzymatic reaction since boiled cells do not exhibit either the redox activity or the ability to release protons. Ferricyanide is an electron aeceptor to which the membranes are impermeable (Craig et el. , 1984) ; thus the reduction of this compound occurs in the extracytoplasmic compartment or in the incubation medium. This process is not due to reducing agents released by the cells since by preincubating cells in the reaction medium, removing them and then adding ferricyanide to the medium, the rate of chemical reduction does not exceed 1 % of that with cells present. The major reducing activity is therefore provided by transmembrane electron transport. /~

pH

A420

6

O.1

0.5 I

5

I 30

Time ( m i n ) Fig.

2 : Stimulation o f H+ e x t r u s i o n by e t h a n o l Cells previously treated 12 h with 3 mM SHAM were incubated for I h without alcohol ( e--e ), with 1 % alcohol ( • • ) or with 1 % alcohol in the presence of 10 mM pyrazole ( G---A ). The reaction was started by adding 5 mM K3Fe(CN) 6

implicated in other systems as the electron donor (Federiea and Giartosio, 1983 ; Chalmers et el. , 1984 ; Lin, 1984 ; Rubinstein et el. , 1984). To test this hypothesis, cells previously treated by SHAM (3 mM) were incubated during i h o u r with ethanol (i %) to increase the production of NADH via alcohol dehydrogenase. When ferricyanide was added to the solution, a stimulation of the excretion of protons was observed (Fig. 2). This alcohol-linked H extrusion is inhibited by pyrazole (Fig. 2), an inhibitor of alcohol dehydrogenase (Chalmers and Coleman, 1983). These data are consistent with the role of endogenous NADH as a substrate for the redox pump. However, in contrast to other results (Rubinstein et el. , 1984) intact C. roseus cells were found not to oxidize exogenous NADH. The reduction of ferricyanide in these conditions was not stimulated by NADH. Nevertheless it can be demonstrated that NADH is a direct substrate for the reaction using a microsomal fraction isolated from these cells ; no ferricyanide reduction occurs until NADH is added to the membrane suspension (Belkoura, unpublished results~. F r o m these data, we can conclude that the

pH 6.5

I 20

I 40

I 60

2mM .ON I O25mMCiC 2

,i 80

Time (rain) Fig. I : Kinetics of FeCN reduction and proton extrusion by C. roseus cells. The pH changes of the medium • • and the redox activity • '• were measured as described in Materials and Methods. The reaction was started with 5 mM K_Fe(CN)~. The pH of the medium without FeCN does n~t chan~e significantly during the time of the experiment. KCN has little effect on the reduction of ferricyanide or the release of protons in C. roseus cells. In contrast SHAM, a well known inhibitor of the alternative pathway, greatly inhibits both processes. So far, the effect of SHAM was tentatively attributed to a decrease in the intracellular NADH concentration, which has been

6,0

5.5

I 15

I 30

Time ( m i n ) Fig. 3 : FeCN-dependent proton extrusion from pro~oplasts and stimulation of the activity with Ca~"[ Proton extrusion was measured by the pH.changes as described in Materials and Methods, with I ml of protoplast suspension (3.10- protoplasts)

313 Table I : Effect of Ca 2+ on the redox activity in isolated protoplasts

Ferricyanide Compounds added

None

reduction

F~CN reduced nmol.h--.10 -v protoplasts

(control)

Change

(%)

400

EGTA

200

- 50

EGTA + CaCI 2

612

+ 53

44

- 89

328

- 18

A23 187 + EGTA A23 187 + EGTA + CaCI 2

The intracellular Ca 2+ concentration is modulated by adding I mM EGTA in the presence or absence of 5 rmM CaC12 and 15 ~M A23 187

of C. roseus cells is impermeable to plasmalemma NADH and that its site of action is localized inside the cells. NADH is not specific as electron donor ; with a microsomal fraction we have observed that NADPH can also play this role but its efficiency is less important, about 30 % compared to NADH. Redox activity and H + extrusion can be detected with ferricyanide using cells or intact protoplasts (Fig. 3). The acidification ~bserved is due to the action of ferricyanide since K , its counterion, given as 6 mM KCL has no effect on the process. This observation provides evidence for the location of

the proton-linked redox activity at the plasmalemma~ rather than at the cell wall. Stimulation of the redox pump by cations with cells or intact protoplasts, the rate of ferrieyanide reduction is strongly inhibited by the addition of the divalent ion chelator EGTA (I mM), and this inhibition c~ be reversed by adding saturating levels of Ca2+ (5 mM) (Table I). The promoting effect of Ca not only acts on the redox activity (Table i) but also on the ferricyanide-dependent proton extrusion (Fig. 3).

Table 2 : Effect of various cations on the redox activity and H ÷ extrusion in isolated protoplasts from C. roseus

Proton extrusion

Redox activity

Salts added

None

(control)

FeCN re_~uced ~nol.h per 10--protoplasts

Change (%) induced by cations

396

~p~.h -I per 10 protoplasts

Change (%) induced by cations

0.36

+ CaCI 2 I mM

630

+ 59

0.52

+

+ MgCI 2 1 mM

498

+ 18

0.42

+ 17

+ MnCI 2 1 mM

84

- 78

nd

+ ZnCI 2 I mM

394

0

nd

+ LaCI 3 I mM

594

+ 50

0.50

+ AIc13 1 mM

492

+ 24

nd

+ KC1 100 mM

380

-

4

nd

+ Nail 100 mM

385

-

3

nd

The the and the

+

45

39

reaction medium was the same as described in Materials and Methods plus indicated concentrations of salts. The different assays were replicated the experiments repeated at least two different times. The data represent results of a typical experiment, nd : not determined

314 Table 3 : Effect oT the net surface charge on the stimulation of ferricyanide reduction by I mM LaC13

Preincubation with CP +

% stimulation of the redox activity by I mM LaCI.

None (control)

CP + 5 ~M

CP + 10 ~M

CP + 50 ~M

41

25

6

0

3 Proto~lasts (106 .ml -I) were preincubated 10 mins with various concentrations of CP- and the reaction was started by I mM K3Fe (CN} 6

The ability to increase redox-linked proton translocatlon is not specific to e a l ~ u m a~+shown in Table ~ Among the cations teste~+Mg--, Laand AI ~- were also active ; Mn-- inhibite~ th~ reaction and the monovalent cations K~ and Na had no effect. 2+ By modifying the Ca gradient across the plasmalemma with the Ca 2+ i°n°~h°rehn we_÷were able to demonstrate t at t form of Ca Z is the intraeellular one. When the protoplast suspension was incubated with the io~$phore and EGTA (low level of cytoplasmic Ca- ) the redox activity decreased. The initial activity was ~$rtially recovered by incubation with additional Ca (Table I). Nevertheless, a plasmalemma-localized reaction is suggested by the msrked stimulation of the redox activity and of H extrusion by lanthanum (Table 2), a non-permeating carson (Rubinstein et al. , 1984). In the case of Ca- , these results ind-ic-~e that it interacts with the internal face of the membrane. A striking stimulation of the redox activity by multivalent cations has already been reported (Federico and Giartosio, 1983 ; Rubinstein et al. , 1984). This general response to cations has been attributed to a "screening" effect, by neutralization of negative surface charges allowing the negatively charged redox agentAto approach the plasmamembrane (Federico and Giartosio , 1983 ; Rubinstein et al. , 1984). In contrast to these findings, a relative specificity of the cations used exists in +our cases Moreover, monovalent cations such as K and Na ~ not stimulate the redox activity and Mn i s even an inhibitor (Table 2). Finally, relatively low concentrations of cations are required ; for Ca 2+ a promoting effect i s seen for concentrations above 0.5 mM and saturation is observed after I0 mM. Thus it appears that cations do not act via an unspecific electrostatic screening effect in C. roseus The surface charge density of the membrane does seem however to play an indirect part in the stimulation of the redox activity. We modified the net charge of the membrane to more positive values by incubating ~rotoplasts with various concentrations of CP , an cationic surfactant (Thibaud et al. , 1983). In these conditions (Table 3) the stimulation of the redox activity by lanthanum decreases _ with the concentration of CP • In contrast, SDS an anionic surfactant

has no effect (results not shown). From these data, one can postulate that the activation process could depend on the attractive effect of the surface charge which in turn affects the concentration of cations in the vfcinity of the membrane. Depending on their specific affinity, cation binding to the plasmalemma might alter the conformation of the redox enzyme in such a way that it interacts more efficiently or not with the substrate. More investigations are needed, nevertheless, to confirm this last hypothesis. In conclusion, the data in this work show that a redox system localized at the plasmalemma is present in Catharanthus roseus cells. One of the functions of such a redox system is thought to be that of a diaphorase, facilitating the uptake of Fe by the reduction of Fe 3+ (Sijmons and Bienfait, 1983). Our results, like others (Lin, 1984 ; Sijmons et al. 1984) introduce another possible function of this redox chain which could serve as an adjunct or alternative to ATPase to drive protons out of the cells.

REFERENCES Barr R., S t o n e B., Craig T.A., Crane F.L. (1985) Biochem. Biophys. Res. Comm. 126 : 262-268 Chalmers J.D.C., Coleman J.O.D. (1983) Biochem. Int. 7 : 785-791 Craig T.A, Crane F.L., Misra P.C., Barr R. (1984) Plant Sci. Lett. 35 : 11-17 Federlco R., Giartosio C.E. (1983) Plant Physiol. 73 : 182-185 Lin W. (1984) Plant Physiol. 74 : 219-222 Marigo G., Delorme Y.M., LUttge U., Boudet A.M. (1983) Physiol. V~g. 21 : 1135-1144 Marigo G., Bouyssou H., Belkoura M. (1985) Plant Science 39 : 97-103 Rubinstein B., Stern A.I., Stout R.G. (1984) Plant Physiol. 76 : 386-391 Sijmons P.C., Bienfait H.F. (1983) Physiol. Plant. 59 : 409-415 Sijmons P.C,, Lanfermeljer F.C., De Boer A.H., Prins H.B.A., Bienfait H.F. (1984) Plant Physiol. 76 : 943-946 Spanswick R.M. (1981) Ann. Rev. Plant Physiol. 32 : 267-279 Thibaud J.B., Romieu C., Gibrat R., Grouzls J.P., Grlgnon C. (1984) Z. Pflanzenphysiol. 114 : 207-213

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells.

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with pr...
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