Plasma Membrane H+ATPase in Maize Roots lnduced for N 0,- U ptake

Plant Physiol. (1 995) 109: 1277-1 283

Plasma Membrane H+-ATPase in Maize Roots lnduced for N 0,- U ptake'

Simonetta Santi, Geraldine Locci, Roberto Pinton, Stefano Cesco, and Zeno Varanini*

Dipartimento d i Produzione Vegetale e Tecnologie Agrarie, University of Udine, Via Delle Scienze 208, 1-331 00 Udine, ltaly

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO,- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO,- or 1.5 mM SO,-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-1 00%) in vesicles isolated from NO,--induced roots. K,,, for Mg:ATP and p H dependency were not influenced by NO,- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO,--induced roots was not affected by 1 O to 150 mM NO,- or CI-. On the other hand, kinetics of NO,-- or CI--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. lmmunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state leve1 of the enzyme in plasma membrane vesicles isolated from NO,--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO,-.

Unlike most high-affinity anion transport systems, which are typically repressible, NO,- uptake is a substrate-inducible process (Clarkson and Lüttge, 1991). Full transporta- tion capacity of this ion can be reached only after contact between the root apparatus and NO,-. Plants that have never been exposed to NO,- take up this ion extremely slowly, and only after root-ion contact can an increase of the uptake be observed. Maximum rates are reached after 3 to 12 h; trends can vary according to experimental conditions and previous nutritional history. The inducible transport system shows Michaelis-Menten saturation and high affinity toward NO,-, and its activity depends on AvH+ components, metabolic energy supply, and plant nitrogen status (Siddiqi et al., 1990; Ullrich, 1992).

NO,- transport from the rhizosphere to the cytoplasm of root cortex cells is thermodynamically disfavored in terms of both electric potential gradient (negative inside the cell) and chemical potential gradient. In fact, NO,- concentration in the soil solution, which is subject to relevant fluc- tuations (Barraclough, 1989), usually ranges from 0.1 to 5

This research was supported by a grant from Italian Ministero Universita e Ricerca Scientifica e Tecnologica (40%) to Z.V.

* Corresponding author; fax 432-558603.

mM (Novoa and Loomis, 1981; Barber, 1984). NO,- concentration in the cytoplasm of root cells exposed to NO,- has recently been estimated to range from 5 to 30 mM (Zhen et al., 1991; King et al., 1992). It is therefore clear that, except under certain conditions (King et al., 1992), NO,- uptake requires energy, presumably as a proton-driving force.

According to results recently acquired in maize (Zea rnays L.) roots using electrophysiological methods (McClure et al., 1990a, 1990b), a direct link between the hyperpolarization caused by NO,- and the activity of the plasma membrane H+-ATPase has been suggested. In particular, a H+ / NO,- symport system seems to operate at the plasma membrane with a Ht/NO3- flux stoichiometry >1, whose activity would cause the stimulation of the H+-ATPase. Moreover, the presence of a H+/NO,- symport carrier on plasma membrane vesicles isolated from maize roots, which can be modulated in accordance with the NO,- status of root cells, has been demonstrated (Ruiz-Cristin and Briskin, 1991; Lu and Briskin, 1993). These observa- tions suggest a link between NO,- transport and the activity of plasma membrane H+-ATPase, especially under conditions of induced NO,- uptake. In addition, as pro- posed by Matzke and Mengel (1993), H+-ATPase could be involved in the reabsorption of anions flowing through the plasma membrane into the apoplast of root cells, a condition that could be enhanced in roots with increased NO,- absorption. So far, no evidence of a direct relationship has been produced for changes of plasma membrane H+- ATPase activity in roots induced for NO,- transport.

In this work, the H+-ATPase activity in plasma membrane vesicles isolated from maize roots treated for 24 h with or without NO,- was studied. The data obtained indicate that ATP-dependent proton pumping and phos- phohydrolytic activity were increased in NO,--induced roots. Results obtained by immunoblotting show that the increase of plasma membrane H'-ATPase activity can be at least partly explained by an enhanced steady-state amount of H+-ATPase within the plasma membrane of maize seedling roots.

Abbreviations: A.O., acridine orange; BTP, 1,3-bis-tris(hy- droxymethy1)propane; AkH+, proton electrochemical gradient; ApH, transmembrane pH difference; K-IDA, potassium iminodi- acetate; TBS, Tris-buffered saline; t,,,, half-time.

1277 www.plant.org on July 6, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1995 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org/

http://www.plant.org

1278 Santi et al Plant Physiol. Vol. 109, 1995

MATERIALS AND METHODS

Plant Crowth

Maize (Zea mays L. cv Eva, Pioneer Hibred Italia SpA) seeds, previously steeped in water for 24 h, were germi- nated over deionized, aerated water in a humid chamber (90% RH) in the dark at 27°C. After 48 h, one-half of the seedlings were transferred into an aerated solution of 1.5 mM CaSO, (control plants). The remaining seedlings were put into an aerated solution of 0.75 mM Ca(NO,), and 0.75 mM CaSO, (induced plants). The seedlings were kept in the dark for another 24 h at 27°C.

In some experiments, roots induced for NO,- uptake were transferred for an additional 24 h to a NO,--free solution (1.5 mM CaSO,) to de-induce NO,- uptake capacity.

NO,- Uptake

To ensure that the inducing treatment was effective, preliminary uptake assays were carried out in vivo. Con- trol and induced roots, quickly rinsed in 1 mM CaSO,, were immersed in aerated solutions of 0.075 mM Ca(NO,), with pH adjusted to 6.0. NO,- depletion from the solutions was measured by spectrophotometer at 207 nm according to Goldsmith et al. (1973).

lsolation of Plasma Membrane Vesicles

Plasma membrane vesicles were isolated from maize roots as previously described by Fisher-Schliebs et al. (1994) with minor changes. Briefly, 10 to 15 g of apical segments (2-3 cm) from induced or control roots were homogenized with a mortar and pestle in a freshly prepared ice-cold medium containing 250 mM SUC, 2 mM MgSO,, 2 mM ATP, 10% (v/v) glycerol, 10 mM glycerol-l- phosphate, 0.16% (w/v) BSA, 2 mM EGTA, 2 mM DTT, 5.7% (w/v) choline-iodide, 1 mM PMSF, and 25 mM BTP titrated to pH 7.6 with Mes. Four milliliters of medium per g fresh weight root tissues were used.

The breis were filtered through four layers of cheese- cloth, and the homogenates were subjected to differential centrifugation steps at 2°C: 1,5008 for 5 min (pellets discarded); 9,800g for 20 min (pellets discarded); 83,400g for 30 min (pellets recovered); and 83,400g for a further 30 min. Microsomes, gently resuspended in 1.2 mL of homogenization medium, were loaded onto a discontinuous Suc gradient made by layering 5 mL of Suc solution (1.13 g cmP3) onto a 9-mL Suc (1.17 g cm-,) cushion, and centri- fuged at 107,600g for 2 h. The Suc solutions were prepared in 5 mM BTP-Mes, pH 7.4, and contained a11 of the pro- tectants present in the homogenization medium.

Vesicles banding at the 1.13/1.17 g cm-, interface were collected, diluted with homogenization medium, and cen- trifuged at 122,400g for 30 min. The pellets, resuspended in a medium containing 250 mM SUC, 10% (v/v) glycerol, 1 mM DTT, and 2 mM BTP titrated to pH 7.0 with Mes, were immediately frozen in liquid N, and stored at -80°C until use.

ATPase Assay

ATP-hydrolyzing activity was measured by determining the release of Pi as described by Forbusch (1983). Assays were carried lout at 38°C in a 0.6-mL reaction volume containing 50 I ~ M Mes-BTP, pH 6.5, 5 mM MgSO,, 0.l mM Na,MoO,, 10 to 15 p g of membrane protein, 150 mM K- IDA, and 50 p~ polyoxyethylene-20-cetyl-ether. Typically, the reaction was started by the addition of 5 mM ATP-BTP (pH 6.5). After 30 min the reaction was stopped by aclding 1 mL of a solution containing 0.6 N HCI, 3% (w/v) SDS, 3% (w/v) ascorbic acid, and 0.5% (w/v) ammonium molyb- date. After 20 min the ATO5 was measured.

In the assays to determine K, and V,,, for Mg:ATP, concentrations of MgSO, and ATP ranged from 0.5 to 10 mM.

To test the composition of membrane vesicles, the ATPase assay was also carried out in the presence or ab- sence of selective inhibitors: 0.1 mM vanadate, 1 mM PJaN,, and 150 mM KNO,, which inhibit plasma membrane, mi- tochondrial, and tonoplast ATPase, respectively. Variations from these assay conditions are indicated in the legertds of tables and figures.

Proton Transport Assay

The formation of a ApH gradient across membrane vesicles was measured as A,,, quenching of A.O. with a Lambda 5 double-beam spectrophotometer (Perkin-Elmer). The assay mixture contained 50 mM Mes-BTP, pH 6.5, 7.5 p~ A.O., and 15 to 20 pg of membrane protein in a final volume of 1.0 mL. After equilibration of the memlbrane vesicles with the reaction medium for 10 min, the reaction was initiated by the addition of Mg:ATP (mixture of MgSO, and ATP-BTP, pH 6.5) to give a final concentration of 5 mM for both compounds. The reaction temperature was 25°C. In the experiments for the determination of the kinetics of NC),- and C1- stimulation of ATP-depeiident Ht-transport, anions were not present in the assay mixture at the beginning of the experiment. In this case the addition of Mg:ATP to energize proton transport determined only a very slight decrease of A.O. A, which after a few minutes reached a steady-state level. Thereafter, appropriate amounts of anion (K+ salt) were added and the decrease in A of the dye followed. Potassium concentration was kept constant (150 mM) by adding K-IDA. Preliminary experiments indicated that IDA-, used as an impermeant anion supplied at different concentrations, did not affect H+- ATPase activity, and essentially no measurable ATP-dependent quenching of A.O. A could be recorded. In the range of anion concentration used (25-150 mM), A.O. A quench, dependent on anion addition (Palmgren, 19911, did not exceed 5% for both NO,- and C1-. As already shown (Fischer-Schliebs et al., 1994), the ATP-depe ndent intravesicular proton accumulation was almost completely abolished by 0.1 mM vanadate, but it was insensitive to NO,- and oligomycin. The ATP-dependent quenching of A.O. was completely reversed by the addition of 8 5 ~ L M

carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 5 PM gramicidin D.

www.plant.org on July 6, 2015 - Published by www.plantphysiol.orgDownloaded from Copyright © 1995 American Society of Plant Biologists. All rights reserved.



H+-ATPase in NO,--1nduced Roots 1279

Measurement of Proton Membrane Permeability

Passive permeability of membrane vesicles to protons was evaluated by measuring the t1,2 of the decrease in the pH gradient after the addition of 10 mM EDTA at the steady state of the intravesicular acidification, according to Lew et al. (1985). Due to the low rate of dissipation of the pH gradient, 8.5 p~ carbonyl cyanide p-(trifluorometh0xy)- phenylhydrazone was added to determine the baseline quench.

Protein Assay

ford (1976), using BSA as standard. Protein contents were determined as described by Brad-

Western Blot Analysis

In membrane vesicle preparations used for western blot analysis, 20 pg/mL chymostatin was added to the homogenization medium to more finely control protease degra- dation. The 122,4008 pellet obtained from centrifugation of vesicles banding at the 1.13/1.17 g cmP3 interface was washed twice by resuspending and pelleting membrane vesicles at 122,400g for 30 min with a solution containing 2 mM BTP-Mes, pH 7.0,250 mM SUC, 10% (w/v) glycerol, and 150 mM KCl. The final pellet was resuspended in an appropriate volume of the following medium: 2 mM BTP- Mes, pH 7.0, 250 mM SUC, 10% (w/v) glycerol, 1 mM DTT, 50 pg/mL chymostatin, and 1 m M PMSF, and thereafter it was immediately frozen in liquid N, and stored at -80°C until use.

Before electrophoresis, samples were solubilized at a ratio of 1:l with SDS loading buffer containing 0.125 M

Tris-HC1, pH 6.8, 10% (w/v) SDS, 0.2 M DTT, 10% (w/v) glycerol, 0.002% (w/v) bromphenol blue, 500 pg/mL chymostatin, and 5 mM PMSF according to Gallagher and Leonard (1987). After incubation at 37°C for 20 min, samples were loaded onto a discontinuous SDS-polyacrylamide gel (3.5% [w/v] acrylamide stacking gel, 9.0% [w/v] acrylamide running gel), according to Laemmli (1970). Re- sulting gels were silver stained (Oakley et al., 1980) according to instructions of the kit (Bio-Rad, Silver Stain kit).

For western blot analysis, after SDS-PAGE proteins were blotted onto nylon membrane filters (Nytran-N, Schleicher & Shuell) overnight at 4°C by a constant current of 40 mA, filters were blocked for 1 h in TBS (20 mM Tris-HCI, pH 7.5, and 150 mM NaCl) containing 5% defatted milk powder, then incubated with antiserum against the SDS-PAGE-separated subunit of maize plasma membrane H+-ATPase (Gallagher, 1985). The antiserum was a kind gift from Prof. R.T. Leonard (University of California, Riverside). Incuba- tion was carried out for 2 h with 1:3000 diluted antiserum in blocking buffer. After washing with TBS containing 0.1% Tween 20, blots were incubated for 1 h with 1:5000 diluted secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG, Sigma). After some washes with TBS containing 0.1% Tween 20, the H+-ATPase subunit was detected by chemiluminescence (ECL Western Blotting Detec- tion System, Amersham) on autoradiography film (Kodak X-Omat AR). Quantification of signal on autoradiography

was obtained by densitometer Ultroscan XL (LKB, Bromma, Sweden).

RESULTS

NO,- Uptake

Roots of 2-d-old maize seedlings placed in contact for 24 h with a 1.5 mM NO,- solution increased the anion uptake rate by about 60% (1.12 versus 1.79 pmol NO,- h-' g-' fresh weight), thus confirming the operation of a NO,-- inducible transport system (Hole et al., 1990).

Plasma Membrane ATPase of NO,--1nduced Roots

Table I shows that plasma membrane vesicles, isolated by discontinuous Suc gradient from homogenates of roots induced for NO,- transport, had values of vanadate-sensitive ATPase specific activity nearly double those of vesicles isolated from roots not exposed to NO,-. A 2-fold increase of ATP hydrolysis was also observed by calculat- ing the enzyme activity on the basis of root fresh weight (not shown). ATPase activity was not significantly influenced by the type of anion used in the assay either in the vesicles isolated from the control roots or in those from roots induced for nitrate uptake. The increase in the enzyme activity was not due to ATPases associated with endomembranes, as proven by the use of selective inhibitors.

Figure 1 shows that the increase in activity of the plasma membrane ATPase depends on root contact with NO,-. In fact, when the seedlings exposed for 1 d to a 1.5 mM NO,- solution were transferred for an additional24 h to a NO,-- free solution (1.5 mM CaSO,), ATPase activity dropped to values below those of the control roots grown in the ab- sence of the anion.

The inducing treatment did not modify the pH dependency of plasma membrane ATPase activity, which showed maximum values at pH 6.1 in both preparations (Fig. 2). V,,, of ATPase activity, calculated in the 0.5 to 10 mM

Mg:ATP concentration range according to the Woolf-Au- gustinsson Hofstee transformation of data, was about 60%

Table 1. Effects o f diagnostic inhibitors on ATP-phosphohydrolyzing activity in plasma membrane vesicles isolated from maize roots treated for 24 h in either 1.5 mM CaSO, (control) or 0.75 m M

CaNO, + 0.75 m M Caso, (NO,--induced)

Assay was performed in the presence of 50 FM polyoxyethylene- 20-cetyl-ether. Data are means from an experiment, run in triplicate, representative of three independent preparations of membrane vesicles. SD did not exceed 5% of the means.

ATP Hvdrolvsis , I

Addition Control NO,--induced

pmol Pi mg- protein h- ' K-IDA 7.2 14.3 K-IDA + vanadate 0.9 0.6 K-IDA + azide 7.0 13.6 KNO, 7.4 13.6 KCI 7.9 14.2




1280 Santi et al. Plant Physiol. Vol. 109, 1995

3" 25 b

h x .* * 20

15

.r( + E l

O E

biz

u a o

2 10

4 õ E 5 3 W

0 Control NO,--Induced

1 1 2

Days of treatment Figure 1. Effect of NO,- treatment of maize roots on ATP-phosphohydrolyzing activity in isolated plasma membrane vesicles. Roots were incubated for 1 d in 1.5 mM Caso, (Control) or 0.75 mM CaNO, + 0.75 mM CaSO, (NO,--1nduced). Thereafter, they were either maintained for 1 more d under the same conditions or transferred to a NO,--free (1.5 mM CaSO,) solution (De-lnduced). Con- ditions of the assay and statistics are as described in Table I. prot, Protein.

higher in vesicles isolated from induced roots than in the controls (15.9 2 1.6 versus 9.9 2 1.1 pmol Pi mg-' protein h-'). On the other hand, K , values were 0.80 2 0.12 mM in vesicles isolated from NO,--induced roots and 0.90 2 0.14 mM in vesicles from control roots.

Figure 3 shows that ATPase activity of vesicles isolated from induced and control maize roots was not affected by NO,- and C1- in the 10 to 150 mM concentration range, thus indicating that the higher ATPase activity observed in plasma membrane vesicles isolated from NO,--induced

.-m NO,'-Induced

O--. Controh. \

\

\/l@\*\J

5 6 7

PH Figure 2. p H dependency of ATP-phosphohydrolyzing activity in plasma membrane vesicles isolated from maize roots incubated for 24 h in 1.5 mM Caso, (Control) or 0.75 mM CaNO, + 0.75 mM CaSO, (NO,--1nduced). Conditions of the assay and statistics are as described in Table I. prot, Protein.

2 2o rControl I NO; -Induced 1

l5I z 5 4 , , , , , , I , , , , , I , I 5

50 100 150 50 100 150 W-

Anion (mM) Figure 3. Effect of CI- and NO,- on ATP-phosphohydrolyzing activity in plasma membrane vesicles isolated from maize roots incubated for 24 h in 1.5 mM CaSO, (Control) or 0.75 mM CaNO, + 0.75 mM CaSO, (NO,---lnduced). Potassium concentration was kept constant at 150 mM by adding appropriate amounts of K-IDA. Other conditions of the assay and statistics are as described in Table I . prot, Protein.

roots was not due to the development of an anion-sensitive enzyme activity.

H+ Transport in Plasma Membranes from NO,--lnduc:ed Roots

The proton-pumping activity of plasma membrane Ht- ATPase was evaluated by measuring the initial rate of A quenching of A.O. in the presence of 150 mM KNO, or KC1 (Table 11). Plasma membrane vesicles isolated from NO,-- induced roots showed a higher rate of ATP-dependent H' accumulation than that of control roots in the presence of both NO,- and C1-. However, the relative increase of intravesicular H+ accumulation was higher with NO,- (+64%) than with C1- (+31%). Table I1 also shows the tl12 of the proton release from membrane vesicles after adding 10 mM EDTA at the steady state of the ATP-dependent ApH formation. The data obtained show that passive permeability to protons was increased (lower tl12) following the root exposure to NO,-. The presence of NO,- or C1- in the reaction mixture used for creating the ApH did not lead

Table II. lnit ial rate of ATP-dependent intravesicular proton accumulation and t,,2 o f pH gradient relaxation in plasma membrã'ne vesicles isolated from maize roots treated for 24 h in either 1.5 mM Caso, (control) or 0.75 mM CaNO, + 0.75 mM Caso, (NO;-- induced)

The rate of A.O. A quench was monitored in the presence of ieither 150 mM KNO, or 150 mM KCI. Data are means from an experiment, run in triplicate, representative of three independent preparations of membrane vesicles. SD did not exceed 5% of the means. Values in Darentheses represent percent of the control.

H+-Transport t,,2 of ApH De<-ay Treatment

NO,- CI- NO,- CI-

AA,,, mg- ' protein min- min Control 0.1 1 (1 00) 0.1 9 (1 00) 62 (1 00) 64 (1 00) NO,--induced 0.18 (164) 0.25 (131) 41 (67) 43 (67)




H+-ATPase in NO3~-lnduced Roots 1281

to significant differences of f , / 2 values either in vesiclesisolated from NO3^-induced roots or in the controls.

The ATP-dependent H+ accumulation was studied atincreasing concentrations (25-150 HIM) of NO3~ or Cl~(Fig. 4). The anions were added in an experimental systemin which the electrical component of AJU,H* was predomi-nant and positive (Lew and Spanswick, 1985), thus provid-ing indirect evidence of the permeability of plasma mem-branes to NO3~ and Q in a direction corresponding invivo to the efflux from the cell (Giannini and Briskin, 1987).Figure 4 shows that in control roots the kinetics of stimu-lation by NO-T and CP of ATP-dependent H+ accumula-tion were considerably different. In fact, NO3~ showed aclear saturable profile, whereas Q stimulation was char-acterized by a pattern with no saturation even at the high-est concentration tested. When the experiment was runusing plasma membrane vesicles isolated from NO3~-in-duced roots, the kinetics of stimulation of intravesicularproton accumulation by anions were different from thoserecorded in vesicles isolated from control roots. In partic-ular, the rate of NO3~-stimulated intravesicular protonaccumulation was clearly higher than that of controls anddid not show saturation within the range of anion concen-trations used. Induction of NO3~ transport by maize rootsalso caused an enhancement of the CF-stimulated protonaccumulation in isolated plasma membrane vesicles, withthe nonsaturating kinetics being still evident.

H+-ATPase Level in NO3~-lnduced Roots

Polyclonal antibodies directed against maize plasmamembrane H+-ATPase were used to compare the amountof H+-ATPase in plasma membrane vesicles isolated fromNO3~-induced and control roots. Silver staining revealed

50 100 150 50 100 150Anion (mM)

Figure 4. Kinetics of CT and NO3~ stimulation of the initial rate ofATP-dependent H+ transport in plasma membrane vesicles isolatedfrom maize roots treated for 24 h in 1.5 mM CaSO4 (Control) or 0.75mM CaNO3 + 0.75 CaSO4 (NO3~-lnduced). Addition of Mg:ATP toenergize proton transport resulted in only a very slight decrease ofA.O. A, which after a few minutes reached a steady-state level. Atthis point appropriate amounts of anion (K+ salt) were added and thedecrease in A of the dye followed. Potassium concentration was keptconstant (1 50 mM) by adding K-IDA. Each point represents the meanof three determinations of three different membrane preparations, sodid not exceed 5% of the means, prot., Protein.

116 .97 -

66

45

Figure 5. Western blot analysis of plasma membrane vesicle pro-teins. Western blotting of plasma membrane fraction proteins fromcontrol (lanes 1) and NOs~-induced (lanes 2) maize roots. A, Silver-stained plasma membrane fraction proteins (3 ;u,g) separated bySDS-PACE. B, Corresponding western blot obtained by using poly-clonal antibodies against maize plasma membrane H+-ATPase and achemiluminescence detection system.

the presence of many polypeptide bands (Fig. 5A). In the100-kD region of the gel, where the subunit of maizeplasma membrane H+-ATPase has been seen to migrate(Gallagher and Leonard, 1987), some narrow bands weredetected. However, no difference in protein pattern andprotein amount between plasma membrane vesicles iso-lated from NO3~-induced and control roots was percepti-ble. Antibodies recognized the H+-ATPase subunit as asingle band of about 100 kD (Fig. 5B) and showed a relativeincrease of H+-ATPase subunit steady-state level in plasmamembrane proteins from NO3~-induced roots. This in-crease could be estimated by densitometric quantificationin an average value of 84% with a SE not higher than 20%of the mean (data not shown); this result is well correlatedwith the relative increase of specific H+-ATPase activity intreated roots.

DISCUSSION

Plasma membrane H+-ATPase is involved in fundamen-tal processes of plant physiology (Serrano, 1989). In fact,this "master enzyme" contributes to the creation and main-tenance of the transmembrane electrochemical gradient fornutrient uptake. Furthermore, it plays a role in the regula-tion of cell turgor and of intracellular and extracellular pH,and it probably underlies control of extension growththrough cell-wall acidification. Due to these crucial func-tions, the operation of plasma membrane H+-ATPase islikely to be tightly regulated at different levels. It has beendemonstrated that plasma membrane H+-ATPase can beactivated by factors like the phytotoxin fusicoccin (de Boeret al., 1989), lysolecithin (Palmgren et al., 1988), trypsin(Palmgren et al., 1990), and, in the case of stomatal cells, bylight (Assmann et al., 1985). De novo synthesis of a high-turnover pool of H+-ATPase molecules was shown to beinduced by the hormone auxin (Hager et al., 1991). Con- www.plant.org on July 6, 2015 - Published by www.plantphysiol.orgDownloaded from

Copyright © 1995 American Society of Plant Biologists. All rights reserved.



1282 Santi et al. Plant Physiol. Vol. 109, 1995

sidering the role of plasma membrane H+-ATPase in plant nutrient acquisition, variation of its activity in relation to plant nutritional status could be expected. Rabotti and Zocchi (1994) showed that Fe-deficient conditions doubled plasma membrane H+-ATPase activity in vesicles isolated from cucumber roots. In Arabidopsis, a differential regulation of the members of the H+-ATPase multigene family has been shown in shoots from plants grown under different developmental or environmental conditions (Harper et al., 1990). However, Samuels et al. (1992) demonstrated that under conditions of reduced K+ availability, which increase K' influx more than 25-fold, there were only minor changes in the expression of plasma membrane H+- ATPase as detected by western blot.

Our data on the effect of NO,- show that the activity of H+-ATPase associated with plasma membrane vesicles isolated from seedling roots treated for 24 h with a solution containing 1.5 mM NO,- is almost 2 times higher than that of control roots (S0,- treated). The higher level of H+- ATPase activity exhibited by NO,--induced roots may be necessary to support the increased rate of NO,- uptake, which is driven by a H+/NO,- symport carrier (Ruiz- Cristin and Briskin, 1991) and thus depends on the availability of Apu,+. McClure et al. (1990a, 1990b) demonstrated that NO,- treatment induced a net hyperpolarization of membrane potential in maize roots, which was attributed to a secondary stimulation of plasma membrane H+- ATPase by the operation of H+/NO,- symport. The data presented here suggest an adaptation of root tissue following the treatment with NO,-, which involves an increased level of plasma membrane H+-ATPase activity. This phe- nomenon appeared to be dependent on the exposure to NO,-. In fact, the H+-ATPase activity of NO,--induced seedling roots transferred for 24 h to a NO,--free solution was similar to that of the control.

The biochemical characteristics of enzyme activities isolated from both control and NO,--induced roots were similar and in good agreement with the features usually displayed by plant plasma membrane H + -ATPase. Fur- thermore, no difference was found in the values of pH optimum, K,, or sensitivity to anions.

Together with the increase in ATP phosphohydrolyzing activity in vesicles from NO,--induced roots, an enhancement of ATP-dependent proton translocating component was recorded, indicating that both functions of H'-ATPase were increased by the exposure of roots to NO,-. The effect on proton transport was more evident when the assay was performed in the presence of NO,- rather than C1F. The experiment, run by starting the proton-pumping reaction with the addition of NO,- and C1- at increasing concentrations, can be useful to get information on the permeability of plasma membrane vesicles to these anions and on the kinetic properties of their transport across the plasma membrane in a direction corresponding in vivo to the efflux from the cell (Giannini and Briskin, 1987). By using this approach, Varanini et al. (1995) showed that transport of NO,- and C1- in plasma membrane vesicles isolated from maize roots not exposed to NO,- could be mediated by proteinaceous structures possessing different kinetic

properties. In fact, the kinetics of stimulation of H+ transport by anions showed a saturable profile for NO,- iand a biphasic pattern for C1-. However, in vesicles obt,ained from NO,--induced roots, the kinetics of NO,-- and C - - stimulated H'-accumulation had a similar pattern, with apparently no saturation for either anion in the concentration range used (25-150 mM). The changes in the kinetics of anion-stimulated H+ transport, which appeared to be more evident for N-O,- than for C1-, suggest that in hJO,-- induced roots, besides an increase in plasma memlbrane H+-ATPase activity, modifications in the operatiori and possibly the regulation of the mechanisms involved in NO,- transport across the plasma membrane take place. Due to the inside-out orientation of plasma mem'brane vesicles competent for proton transport, the obtained results could support the idea of an involvement of NO3- efflux in the regulation of NO,- uptake.

Results obtained by western blot analysis showecl that the relative increase of plasma membrane H+-ATPase specific activity in NO,--induced roots can be at least partly explained by an increased steady-state level of this enzyme within the plasma membrane of maize seedling roots. In this regard, it is worthy to note that so far an increased level of plasma membrane H+-ATPase has been demonstrated only in the case of maize coleoptiles treated with auxin (Hager et al., 1991). De novo synthesis of plasma membrane H* -ATPase in NO,--induced roots might well be necessary to sustain the increased fluxes of NO,- across the plasma membrane (McClure et al., 1990a, 1990b).

Severa1 isoforms of plasma membrane H+-ATPase have been identified (Moriau et al., 1993) that show different biochemical characteristics (Palmgren and Christensen, 1994). Increase in plasma membrane H+-ATPase activity following NO,- treatment could involve one or more specific isoforms. Although our experiments do not allow us to draw conclusions about this, it is noteworthy that in Nico- tiann plumbagiizifolia a single plasma membrane H+-AI'Pase isoform is expressed in cells and tissues involved in iactive nutrient transport (Michelet et al., 1994).

In conclusion, our results clearly show that exposure of maize roots to NO,-, a condition that induces a higher rate of net NO,- uptake, causes a significant increase in plasma membrane H+-ATPase, supporting the idea of an involvement of this enzyme in the responses of roots to fluctuating externa1 NO,- concentrations.

ACKNOWLEDCMENT

The authors thank Prof. R.T. Leonard (University of California, Riverside) for the generous gift of polyclonal antibodies a gainst maize plasma membrane H+-ATPase.

Received July 13, 1995; accepted September 14, 1995. Copyright Clearance Center: 0032-0889/95/l09/1277/07.

LITERATURE ClTED

Assmann S, Simoncini L, Schroeder J (1985) Blue light achates electrogenic ion pumping in guard cell protoplasts of Vicia faba. Nature 318: 285-287




H+-ATPase in NO,--lnduced Roots 1283

Barber SA (1984) Soil Nutrient Bioavailability: A Mechanistic Ap- proach. John Wiley, New York

Barraclough PB (1989) Root growth, macronutrient uptake dy- namics and soil fertility requirements of a high-yielding winter oilseed rape crop. Plant Soil 119: 59-70

Bradford MM (1976) A rapid and sensitive method of quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7 2 248-254

Clarkson DT, Liittge U (1991) Mineral nutrition: inducible and repressible nutrient transport systems. In H-D Behnke, K Esser, K Kubitzki, M Runge, H Ziegler, eds, Progress in Botany, Vol52. Springer Verlag, Berlin, pp 61-83

de Boer AH, Watson BA, Cleland RE (1989) Purification and identification of the fusicoccin binding protein from oat root plasma membrane. Plant Physiol 89: 250-259

Fischer-Schliebs E, Varanini 2, Lüttge U (1994) Isolation of Hf- transport-competent plasma membrane vesicles from corn roots by discontinuous sucrose gradient centrifugation: effect of membrane protectant agents. J Plant Physiol 144: 505-512

Forbusch B (1983) Assay of the Na+K+-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulphate buffered with bovine serum albumin. Anal Biochem 128: 159-163

Gallagher SR (1985) Purification and electrophoretic analysis of the plant plasma membrane ATPase. PhD thesis, University of California, Riverside

Gallagher SR, Leonard RT (1987) Electrophoretic characterization of a detergent-induced plasma membrane fraction from maize roots. Plant Physiol 83: 265-271

Giannini JL, Briskin DP (1987) Proton transport in plasma membrane and tonoplast vesicles from red beet (Betu vulgaris L.) storage tissue. A comparative study of ion effects on ApH and AT. Plant Physiol84: 613-618

Goldsmith J, Livoni JP, Norberg CL, Segel IH (1973) Regulation of nitrate reductase in Penicillium ckrysogenus by ammonium ion. Plant Physiol 52: 362-367

Hager A, Debus G, Edel HG, Stransky H, Serrano R (1991) Auxin induces exocytosis and the rapid synthesis of a high-turnover pool of plasma membrane H+-ATPase. Planta 185 527-537

Harper JF, Manney L, De Witt ND, Yoo MH, Sussman MR (1990) The Arubidopsis tkaliana plasma membrane H+-ATPase multigene family. J Biol Chem 265: 13601-13608

Hole DJ, Emran AM, Fares Y, Drew MC (1990) Induction of nitrate transport in maize roots, and kinetics of influx, measured with nitrogen-13. Plant Physiol 93: 642-647

King B, Siddiqi MY, Glass A (1992) Studies of the uptake of nitrate in barley. Estimation of root cytoplasmatic nitrate concentration using nitrate reductase activity: implications for nitrate influx. Plant Physiol 99: 1582-1589

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685

Lew RR, Bushunow N, Spanswick RM (1985) ATP-dependent proton pumping activities of zucchini fruit microsomes. A study of tonoplast and plasma membrane activities. Biochim Biophys Acta 821: 341-347

Lew RR, Spanswick RM (1985) Characterization of anion effects on the nitrate-sensitive ATP-dependent proton pumping activity of soybean (Glycine max L.) seedling root microsomes. Plant Physiol 77: 352-357

Lu Q, Briskin D (1993) Modulation of the maize plasma membrane carrier by NO,-. Phytochemistry 33: 1-8

Matzke H, Mengel K (1993) Importance of plasmalemma ATPase in the retention and exclusion of inorganic ions. Z Pflanzener- naehr Bodenkd 156: 515-519

McClure PR, Kochian LV, Spanswick RM, Shaff JE (1990a) Evi- dente for cotransport of nitrate and protons in maize roots. Effect of nitrate in the membrane potential. Plant Physiol 93:

McClure PR, Kochian LV, Spanswick RM, Shaff JE (1990b) Evi- dente for cotransport of nitrate and protons in maize roots. Measurement of NO,- and Hf fluxes with ion-selective microelectrodes. Plant Physiol 93: 290-294

Michelet B, Lukaszewicz M, Dupriez V, Boutry M (1994) A plant plasma membrane proton-ATPase gene is regulated by development and environment and shows signs of a translational regulation. Plant Cell 6 1375-1389

Moriau L, Bogaerts P, Jonniaux JL, Boutry M (1993) Identification and characterization of a second plasma membrane H+-ATPase gene subfamily in Nicotiann plumbaginifolia. Plant Mo1 Biol 21:

Novoa R, Loomis RS (1981) Nitrogen and plant production. Plant Soil58 177-204

Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasen- sitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105 361-363

Palmgren MG (1991) Acridine orange as a probe for measuring pH gradient across membrane: mechanism and limitations. Anal Biochem 192: 316-321

Palmgren MG, Christensen G (1994) Functional comparison between plant plasma membrane Hf-ATPase isoforms expressed in yeast. J Biol Chem 269: 3027-3033

Palmgren MG, Larsson C, Sommarin M (1990) Proteolytic activa- tion of the plasma membrane H+-ATPase by remova1 of a ter- minal segment. J Biol Chem 265: 13423-13426

Palmgren MG, Sommarin M, Kerskov P, Jorgensen PL (1988) Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acid and phospholipase A,. Physiol Plant 74: 11-19

Rabotti G, Zocchi G (1994) Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots. Physiol Plant 90: 779-785

Ruiz-Cristin J, Briskin DP (1991) Characterization of a Hf/N03- symport associated with plasma membrane vesicles of maize roots using 36C103- as a radiotracer analog. Arch Biochem Bio- phys 285 74-82

Samuels AL, Fernando M, Glass ADM (1992) Immunofluorescent localization of plasma membrane H+-ATPase in barley roots and effects of K nutrition. Plant Physiol 99: 1509-1514

Serrano R (1989) Structure and function of plasma membrane ATPase. Annu Rev Plant Physiol Plant Mo1 Biol 40: 61-94

Siddiqi MY, Glass ADM, Ruth TJ, Rufty TW Jr (1990) Studies of the uptake of nitrate in barley. Kinetics of ',NO,- influx. Plant Physiol93: 1426-1432

Ullrich WR (1992) Transport of nitrate and ammonium through plant membranes. In K Mengel, DJ Pilbeam, eds, Nitrogen Me- tabolism of Plants. Clarendon Press, Oxford, UK, pp 122-135

Varanini 2, De Biasi MG, Pinton R (1995) Effect of NO,-, C1- and DIDS on H+-ATPase of plasma membrane vesicles isolated from corn roots. J Plant Physiol 146 423428

Zhen R-G, Koyro H-W, Leigh RA, Tomos AD, Miller AJ (1991) Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling. Planta 185: 356-361

281-289

955-963




Plasma Membrane H+ATPase in Maize Roots lnduced for N 0,- U ptake

Documents

Transcript of Plasma Membrane H+ATPase in Maize Roots lnduced for N 0,- U ptake