Is sodium proton antiport ubiquitous in plant cells?

J.PlantPhysiol. Vol. 137.pp. 180-183(1990)

Is Sodium Proton Antiport Ubiquitous in Plant Cells? HARTWIG MENNEN l , 2, BENJAMIN ]ACOByl*,

and H. MARSCHNER2

1 Department of Agricultural Botany, The Hebrew University of Jerusalem, Rehovot P.O. Box 12, 76100 Israel 2 Institut flir Pflanzenernahrung, Universitat Hohenheim, 7000 Stuttgart 70, FRG * To whom correspondence should be addressed.

Received May 7, 1990 . Accepted July 12, 1990

Summary Excised roots or storage tissue slices from 16 crop plant species were screened for the presence of a Na +/H + antiporter at the plasma membrane and tonoplast of their cells. The pH-gradient dependent decrease of Na+ uptake by ATP-depleted tissues served as an indication for such antiport at the plasma membrane. Metabolic-energy dependent uptake by the tissues, in the presence of excess K +, indicated the functioning of Na+/H+ antiport at the tonoplast. Evidence for Na+/H+ antiport was found in four species and for its absence in five species. In seven species the evidence was not unequivocal. It was concluded that the presence of a Na +/H+ antiporter is not an ubiquitous characteristic of plant cells.

Key words: Crop plants; sodium; Na+ iH+ antiport; Cl- -H+ symport; metabolic uptake. Abbreviation: FCCP

=

carbonylcyanide p-(trifluoromethoxy)-phenyl hydrazone.

Introduction Sodium/proton (Na+/H+) antiport is postulated to function at the plasma membrane and tonoplast of plant cells. It should transport Na+ out of the cytoplasm to the apoplast, or into the vacuole, or both. Hitherto the evidence for this assumption is limited to a few plants species, namely Hor· deum vulgare roots at the plasma membrane (Ratner and J acoby, 1976; Colombo et al., 1979; Jeschke, 1980) and at the tonoplast (Garbarino and DuPont, 1988, 1989; Fan et aI., 1989), Beta vulgaris at the plasma membrane Gacoby and Teomi, 1988) and at the tonoplast (Blumwald and Poole, 1985), Atriplex nummularia roots at the plasma membrane (Braun et al., 1988), Cataranthus rosea at the tonoplast (Guern et al., 1989), Chara corallina at the plasma membrane (Clint and MacRobbie, 1987) and Dunaliella salina at the plasma membrane (Katz et aI., 1986, 1989). Evidence for the activity of this antiport could not be found in some other plant species, namely Zea mays (Cheeseman, 1982; Jacoby and Rudich, 1985; Jacoby and Teomi, 1988) and Phaseolus vulgaris Gacoby and Ratner, 1974). These negative results evoke the question whether the N a +/H + antiporter is in© 1990 by Gustav Fischer Verlag, Stuttgart

deed widely distributed in plants; has it been lost during the evolution of some very salt sensitive plant species such as Z. mays and P. vulgaris, or is it also missing in some relatively salt-resistant glycophytes? In the latter case some other major mechanism for the exclusion of sodium from the cytoplasm should have evolved. Recently, an assay for the assessment of Na +/H + antiport at the plasma membrane of intact plant tissues was described Gacoby and Teomi, 1988). This assay measures the effect of an artificial pH gradient on net 22Na + uptake by ATP depleted tissues. Uptake at pH 7.0 is compared to that at pH 3.9, obtained by acidification with either H 2S04 or butyric acid. With H 2S04 not only acidification of the external pH is obtained, but also a transmembrane pH gradient (acid outside). On the other hand, the weak butyric acid permeates the plasma membrane in its undissociated form (Reid et aI., 1989), and is not expected to establish a pH gradient, or at least to establish a significantly smaller one. A pH gradient should support outward directed Na+ transport by N a +/H + antiport at the plasma membrane, and result in decreased net 22Na + uptake. An analogous effect of these acids on CI- - H + symport was shown Gacoby and Rudich, 1980;

181

Sodium proton Antiport in plant cells

Jacoby and Teomi, 1988}. This assay was now employed to screen 16 crop plant species for Na +/H + antiport at the plasma membrane of their root or storage tissue cells. Sodium influx at the plasma membrane of plant cells is apparently non-metabolic (metabolic-energy dependent) in the presence of excess K + (Ratner and Jacoby, 1976; Jacoby and Hanson, 1985; Jacoby and Rudich, 1985). The strong suppression of Na+ uptake by K+ was already shown by Rains and Epstein (1967). Hence, whenever metabolic Na+ uptake into plant cells occurs in the presence of excess K +, it appears to depend on Na +/H + antiport at the tonoplast (Ratner and Jacoby, 1976). Metabolic Na+ uptake served in this survey as an indicator for the activity of aNa +/H + antiporter at the tonoplast.

Materials and Methods Plants Cultivars of the following plant species were used:

Species A vena sativa L. Beta vulgaris L. Brassica oleracea L. Chloris guayana Kth. Gossipium hisutum L. Helianthus annuus L. Hordeum vulgare L. Lycopersicum esculentum Mill. Panicum maximum Jacq. Phaseolus vulgaris L. Sorghum bicolor L. Moench Sorghum sudanense (Piper) Stapf Ricinus communis L. Triticum aestivum L. Vigna radiata L. Zea mays L.

Cultivar Saia 6 Early Flat Egyptian Green Express Samford Alcala SJ22 Orbit Ruth Holit Giant Bronco Hazera 610 Hazera Hazera 22 Inia 66 (unknown) Gibli

Seeds were germinated on 0.2 mM CaS04 and grown in the dark until the roots were about 50 mm long. Roots were then excised and washed for 4 h in 0.2 mM CaS04 at 25°C and used for the measurement of Na + and Cl- fluxes. B. vulgaris storage tissue slices were prepared as described before Gacoby and Teomi, 1988) and also used for flux measurements (net uptake).

Ion Fluxes Previously described assays were employed to assess Na+ /H+ antiport and CJ- - H+ symport at the plasma membranes of tissues Gacoby and Rudich, 1980; Jacoby and Teomi, 1988). In brief, excised and washed roots, or aged beet slices, were anaerobically preincubated for 10 min at 30°C in 0.2 mM CaS04 and 1.0 mM K 2S0 4 at pH 7.0 to deplete ATP. Double labelled 22Na36 Cl was then added to a final concentration of 0.1 mM without, or with H 2S0 4 or nbutyric acid to adjust the pH to 3.9, and anaerobic incubation was continued for a further 10 min. The tissue was washed in 10 mM 4 at 0 °C to remove exchangeable ions and radioassayed. To assess the presence of Na+ /H+ antiport at the tonoplast, metabolic Na + absorption was measured in the presence of excess K + . The tissue was aerobically incubated for 30 min at 30°C in 0.2 mM 4, and 0.1 mM 22Na 36 CJ without, or with 1.0 mM K 2S0 4 and 5 I'M FCCP.

caSo caSo

Results Chloride uptake from 22Na36Cl, by ATP depleted tissues of all the screened species, was significantly enhanced at pH 3.9 when adjusted with H 2S04 • This enhancement was completely or partially abolished when pH 3.9 was adjusted with butyric acid. The differences between the H 2S04 and butyric acid treatments were highly significant (p < 0.01) for all species. Similar results were previously obtained for barley roots, corn roots, and beet slices acoby and Rudich, 1980; Jacoby and Teomi, 1988} and interpreted as an indication for Cl- - H + symport at the plasma membrane. In the present investigation, this response of CI- uptake served as an internal control for the normal function of the investigated tissues and of the assay. The detailed results for CI- uptake are not presented. The effect of low external pH on Na+ uptake by ATP depleted tissues is shown in Table 1. In one species, S. bicolor, N a + uptake was decreased by both acids. In A. sativa, B. vul· garis, H annuus, H vulgare, L. esculentum, and T. aestivum adjustment of pH to 3.9 with H 2S04 significantly decreased Na + uptake, while adjustment to the same pH with butyric acid had no significant effect. In a further eight species, B. ole·

a

racea, G. hirsutum, P. maximum, P. vulgaris, R. communis, S. sudanense, radiata and Z. mays, decrease of external pH to

v.

3.9 did not effect Na + uptake significantly, irrespective of whether pH was decreased with H 2S04 or butyric acid. The protonophore FCCP inhibited aerobic CI- uptake in all tested species (data not presented). The inhibition ranged between 80 % and 98 % in the different species, and again indicated proper function of inhibitor and plant tissues. The effect of FCCP on aerobic Na + uptake by roots or tissue slices from 16 plant species is shown in Table2. Inhibi-

Table 1: 22Na uptake under anoxia at pH 7.0 and pH 3.9 adjusted with H 2S04 or BA (n-butyric acid). Anoxic pretreatment: 10 min in 0.2 mM CaS04, 1.0 mM K2S04 at pH 7.0. NaCI was then added to 0.1 mM without, or with acid to adjust pH to 3.9; anoxic incubation was continued for additional 10 min.

Species

Na+ uptake: nmol·g- 1 ± SE* pH 7.0 pH 3.9 SD** (A) BA(B) H 2S0 4 (C) (B>C) 1.7 tO.2b 3.S±0.2" 3.2tO.3" + 6.S±O.6" 7.8±O.7" 3.3 ±O.6 b + 15 tlA" 12 tOA" 3.StOA" 2AtO.3" 1.ltO.l b + 14 to.s" 13 to.7" 13 t 1.0" 8.8tOA" 6.7tO.6b + 9.stOAb 11 tOA" 13 t 1.0" + 6.2tO.S" 4.9tO.S" 3.0tO.s b + 10 ±0.7" 7.3±0.7 b 8.2±0.9"' b 9.0±O.s" 7.9±0.7" 14 to.7" 23 ± 1.8 b 18 t 104" 8.U 1.3" 2.6tO.Sb 3.3±0.3 b

A vena sativa Beta vulgaris Brassica oleracea Chloris guayana Gossipium hirsutum Helianthus annuus Hordeum vulgare Lycopersicum escul. Panicum maximum Phaseolus vulgaris Ricinus communis Sorghum bicolor Sorghum sudanense 15 t2.7" 15 t2.8" 11 to.9" Triticum aestivum 8.9tO.3" 11 to.9" 6.8±OAb Vigna mungo 7.6tO.6" SAtO.3b 7A±0.3" Zea mays 8.0tO.5" 8.3tO.S" 7.2tO.2"

+

.. Standard error, different superscripts indicate significant difference (p < 0.05, n = 5), .... Significant difference (p < 0.05 = +).

182

HARTWIG MENNEN, BENJAMIN JACOBY, and H. MARSCHNER

Table 2: 22Na + uptake from 0.1 mM NaCl, 0.2 mM caSo4 in the absence or presence of 0.1 mm K2S04 and 5 11M FCCP. -K + Control±SE* (A) 222± 1.0 273±22 48± 3.1 82± 8.2 59± 3.9 96±12 275± 9.0 462±26 60± 4.8 32± 2.8 30± 4.6 176±17 250±22 68±13 11± 0.6 28± 2.3

Species A. sativa B. vulgaris B. okracea C guayana G. hirsutum H. annuus H. vulgare L. esculentum P. maximum P. vulgaris R. communis S. bicolor S. sudanense T. aestivum V.radiata Z mays * Standard error. ** Significant difference (p<0.05 *** Not determined.

=

FCCP±SE (B) 11 ± 1.0 40±1.3 28± 1.4 32±2.6 57±0.1 19± 1.5 25±3.5 18±0.6 15±0.6 28± 1.7 28±2.3 22±1.6 22±1.6 12±0.9 13±1.3 25±0.9 +, n

a

Na +uptake: nmol· g- l. (30 min)-l SD.** (A>B) +

+ + + + + + +

+

+ +

Control±SE (C) 4.2±0.3 13 ±6.1 6.8±0.6 17 ±1.1 33 ±0.6 38 ±4.2 66 ±2.2 15 ±0.9 28 ± 1.7 nd*** nd 68 ±2.2 61 ±0.5 14 ±0.7 nd 28 ± 1.7

+K+ FCCP±SE

(D)

SD. (C>D)

5.7±0.2

2.0±OA

30 ±3.7 17 ±1.4 30 ±1.2 50 ±3.0 · 25 ±0.7 10 ±0.7 22 ±3.1 nd nd 24 ±2.1 7.3±0.5 7.9±OA

nd 30 ±0.7

+

+ +

+ + +

5).

tion of energy metabolism depressed Na + uptake in 11 of these species when K + was absent, but had no effect in G. hirsutum, P. vulgaris, R. communis, V. radiata and Z. mays. Addition of 1.0mM K 2S0 4 drastically decreased Na+ uptake in all those species in which it was inhibited by FCCP in the absence of K +. In the presence of 1.0 mM K 2S04 , FCCP inhibited Na + uptake only in 6 species, namely B. vulgaris, H vulgare, L. esculentum, S. bicolor, S. sudanense and T. aestivum. In an additional experiment G. hirsutum was germinated and grown in 1/10 artificial sea water. This pretreatment did not induce metabolic sodium uptake by aerobic roots, and also not Na+ IH+ antiport in anaerobic ones (not shown). Discussion The best way to establish the presence of a transport system, like a Na +/H+ antiporter, seems to be examination in isolated membrane vesicles and further purification and incorporation into liposomes. This is, however, not appropriate for a survey including many species. Here we adopted an assay developed for assessment of N a + fluxes in intact tissues. In this assay a pH-gradient dependent decrease of net 22Na+ flux into ATP depleted tissue is taken as an indicator for Na+/H+ antiport. As protons may block Na+ channels (Hille, 1984), it had to be assured that decreased net Na+ influx resulted from a pH-gradient dependent enhancement of efflux, and not from a low-pH dependent blockage of influx. This was achieved by comparing the effects of lowering pH with H 2S0 4 an butyric acid and by checking the effect of these acids on CI- uptake by the same tissue samples. Chloride influx into ATP depleted tissues (CI- -H+ symport) was enhanced only by adjusting pH to 3.9 with H 2S0 4

and not with butyric acid, indicating that the latter acid indeed did not form an additional transmembrane pH gradient. Thus, a significantly larger inhibition of Na+ uptake by H 2S04 than by butyric acid was used a criterion for the functioning of Na+/H+ antiport at the plasma membrane. According to this criterion, in seven out of sixteen species evidence for Na +IH + antiport activity at the plasma membrane was found (Table 1). In nine species Na+ IH+ antiport at the plasma membrane was not indicated. With the exception of S. bicolor, in eight of these species Na+ uptake did not decrease at pH 3.9, neither with butyric acid nor H 2S04 (Table 1). The possible function of a Na+ IH+ antiporter at the plasma membrane of roots was previously examined by the effect of external acidification on Na + efflux from preloaded, 1.0 mm long, root tips of H vulgare and Z. mays (Ratner and Jacoby, 1976; Jacoby and Rudich, 1985). The present assay confirms the previous results for these two species, namely presence of Na+ IH + antiport in H vulgare and its absence in Z. mays (Table 1). The assessment of metabolic Na+ transport at the tonoplast of intact tissues is complicated by interaction with K + at the plasma membrane and possibly at the tonoplast as well. In the absence of external K + , N a +uptake may be mediated by the metabolic K +-pathway at the plasma membrane acoby and Hanson, 1985). In Z. mays roots this pathway is blocked by 2.0mM K+ at 10mM external Na+ (a liS K+I Na+ ratio). In the present assay a 100-fold lower Na+ concentration was employed and it was thus assumed that 2.0mM K + (a 20/1 K+/Na+ ratio) would block metabolic Na+ uptake at the plasma membrane. Under such conditions metabolic Na + uptake by the tissues should depend on transport at the tonoplast into the vacuole. However, the absence of metabolic Na + uptake is not a clear indication for the absence of aNa +IH + antiporter at the tonoplast. An ex-

a

Sodium proton Antiport in plant cells ternal high K + IN a + ratio may lead to a similar ratio at the cytosol/tonoplast interface and a too low cytosolic Na + concentration for demonstration of metabolic Na + uptake at the tonoplast. The Na + IK + selectivity of transport at the tonoplast is not well known, although a K + insensitive component of Na+ IH+ antiport was shown in tonoplast vesicles (Blumwald and Poole, 1985; Garbarino and DuPont, 1989). In the absence of external K + , in 11 of the 16 investigated species metabolic Na+ uptake occurred (Table 2). This metabolic Na+ uptake cannot be ascribed to tonoplast transport, because it may depend on metabolic transport at the plasma membrane via the K+-pathway. The lack of metabolic Na+ transport in the remaining five species in the absence of K +, can be interpreted as good evidence for the nonexistence of metabolic Na+ uptake at their tonoplasts. In five additional species (A. sativa, B. oleracea, C. guayana, H annuus, P. maxi· mum) metabolic Na+ uptake was found in the absence of K +, but not in its presence (Table 2). The roots of these plants seem also not to perform metabolic Na+ uptake at the tonoplast, but the evidence for this is not unequivocal. Metabolic Na+ uptake in the presence of K+ was found only in five species (Table2) and indicates the presence of Na +IH+ -antiport at the tonoplast. The results of the present investigation support the assumption that Na + IH+ antiport at the plasma membrane and tonoplast is not an ubiquitous characteristic of plant cells. Evidence for the functioning of Na+/H+ antiport at the plasma membrane as well as at the tonoplast was found for B. vulgaris, H vulgare, L. esculentum and T. aestivum. Good evidence for the absence of this antiport at both membranes was found for G. hirsutum, P. vulgaris, R. communis, V. radiata and Z. mays. For other plant species the data obtained are less conclusive. Additional information is expected from work with membrane vesicles (now under progress). Blumwald and Poole (1987), Garbarino and DuPont (1988, 1989) and Guern et al. (1989) induced Na + IH+ antiport in isolated barley and beet tonoplast-vesicles by a NaCI pretreatment of the tissue before separation of the membranes. In the present investigation with intact barley and beet tissues, such activation was not required. Furthermore, our attempt to induce Na+ IH+ antiport in C. hirsutum by growing the plants in 1/10 seawater (50 mM NaCl) was not successful. Some of the plant species, like C. hirsutum, in which a Na +IH + antiport seems to be absent, are considered rather salt tolerant (Maas and Hoffman, 1977). They should have evolved some mechanism for prevention of Na+ influx to keep cytoplasmic Na + concentration low. Indeed, in G. hir· sutum and all other species for which the nonexistence of Na +IH+ antiport was indicated, aerobic Na + uptake was low even in the absence of K + (Table 2). Acknowledgements This research was supported by a grant of the Land Baden-Wiirttemberg, FRG.

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