Journal of Experimental Botany, Vol. 52, No. 365, pp. 2355-2365,
December 1, 2001
© 2001 Oxford University Press
Original Papers |
Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa
Institut für Botanik, Technische Universität Darmstadt, Fachbereich 10, Schnittspahnstraße 3–5, D-64287 Darmstadt, FRG
Received 29 May 2001; Accepted 20 July 2001
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Abstract |
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The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.
Key words: Immunoprecipitation, salinity, V-ATPase, V-PPase, Suaeda salsa L.
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Introduction |
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The Chenopodiaceae Suaeda salsa L. is native to saline soils of northern China and is adapted to growth in the high salt flooding region. S. salsa is one of the most important halophytes in China. Its seeds contain about 40% oil, rich in unsaturated fatty acids, which is a good food oil and can easily be converted to chemical compounds for industrial use (Zhao, 1998
). S. salsa can tolerate coastal seawater salinity and salinity fluctuations resulting from water evaporation and tidal inundation. Generally, the cytosolic Na+ concentration in both halophytes and non-halophytes does not exceed about 0.15 M, because otherwise a variety of metabolic reactions would be inhibited (Flowers et al., 1986). In contrast to some other halophytic plants, S. salsa does not have salt glands or salt bladders on its leaves. Thus, this plant must compartmentalize the toxic Na+ in the vacuoles. Therefore, membrane-bound transport systems regulating cytosolic ion homeostasis (Na+, K+ and Ca2+) and ion accumulation in the vacuole can be considered of crucial importance for adaptation to saline conditions (Serrano et al., 1999; Hasegawa et al., 2000). The capacity of Na+ transport from the cytoplasm into the vacuole via the tonoplast Na+/H+ antiport (Barkla et al., 1995; Fukuda et al., 1998) is dependent on the activity of V-ATPase (EC 3.6.1.35) and V-PPase (EC 3.6.1.1), which establish an electrochemical H+-gradient across the tonoplast that energizes the transport of Na+ against the concentration gradient (for review see Binzel and Ratajczak, 2001). The effects of salinity on tonoplast proteins have been intensively studied in the halophyte and salt-includer Mesembryanthemum crystallinum. After 8 d of salt treatment, V-ATPase protein amount and activity increase in leaf cells by a factor of 2.5 (Ratajczak et al., 1994), while V-PPase amount and activity decrease (Bremberger and Lüttge, 1992a; Rockel et al., 1994). Recently, the ability of M. crystallinum to tolerate salt was studied in a truly salt-tolerant cell line prepared from leaves of 6-week-old well-watered M. crystallinum plants. These cells showed a salt-induced increase in Na+ transport and V-ATPase activity, while osmotic stress did not induce any changes in V-ATPase activity (Vera-Estrella et al., 1999). In a cell line isolated from the non-halophyte Daucus carota only V-PPase activity was markedly stimulated by salt treatment, while it was unaffected by sorbitol treatment (Colombo and Cerana, 1993). In general, salinity leads to an increase in V-ATPase activity, while it remains constant or decreases in non-halophytes (Binzel and Ratajczak, 2001).
K+ has been considered to play a role in salt toxicity (Läuchli, 1990; Niu et al., 1995). In non-halophytes, Na+ salinity induces a decrease in the K+ content of the tissue, which has been proposed to contribute to salt toxicity which is correlated to a decrease in plant growth (Ben-Hayyim et al., 1987; Nakamura et al., 1990). A lack in K+ nutrition rather than Na+ excess was shown to play a critical role in salt damage of non-halophytes (Zhu et al., 1998). In some halophytes salinity increases the K+ concentration of the tissue. However, even when the tissue K+ content was decreased, plant growth was always significantly promoted by a certain level of salinity (Flowers et al., 1977; Greenway and Munns, 1980).
K+ transport is mediated by K+ channels as well as high affinity K+ transporters, both in the plasmalemma and in the tonoplast of plant cells (Niu et al., 1995; Serrano et al., 1999; Schachtman, 2000). The mechanism of Na+ uptake into plant cells across the plasmalemma is not yet clear. It might be mediated by K+ channels and non-selective cation channels (Amtmann and Sanders, 1999; Schachtman and Liu, 1999; Rubio et al., 1995; Serrano et al., 1999). The extrusion of Na+ from the cytoplasm to the apoplast or the vacuole is mediated by Na+/H+ antiporters located in the plasmalemma or the tonoplast (Hassidim et al., 1990; Garbarino and DuPont, 1988; Barkla et al., 1995; Ballesteros et al., 1997; Fukuda et al., 1998; Morsomme and Boutry, 2000).
Taken together, Na+ and K+ transport across plant membranes depends directly or indirectly on electrochemical gradients across these membranes established by primary active proton pumps, i.e. the P-type H+-ATPase (P-ATPase) at the plasmalemma and the V-ATPase and the V-PPase at the tonoplast (Sze et al., 1999; Maeshima, 2000; Ratajczak, 2000). The aim of the present study was to investigate the effects of NaCl, KCl and osmotic stress on growth parameters and vacuolar primary-active proton pumps of the halophyte S. salsa, which is a C3-plant as determined by microscopic inspection of leaf anatomy, and, in contrast to M. crystallinum, is not able to switch photosynthetic metabolism to crassulacean acid metabolism (CAM). In addition to the determination of V-ATPase and V-PPase activity and protein amount, the subunit composition of the V-ATPase was studied in preparations from plants grown under different conditions in order to correlate changes in enzyme activity with structural modifications.
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Materials and methods |
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Plant material
Seeds of Suaeda salsa L. were collected on the salt flats at the shore of the Yellow Sea, China. Seeds were soaked in tap water for 3 h and germinated in sand. Seedlings were raised in sand for 6 weeks and irrigated daily with full-strength Hoagland and Snyder solution (Hewitt, 1963
). Subsequently, one batch of seedlings was further irrigated with Hoagland solution (controls), while other batches were subjected to salt treatment in a daily increment of 0.05 M NaCl or KCl or 0.016 M PEG 4000 (iso-osmotic to 0.05 M NaCl) to the final concentrations as indicated in the tables and figures. The seedlings were grown for further 16 d at the following conditions: temperature 28/20 °C, RH 70/80% (day/night) at a photoperiod of 16 h d-1 provided by supplementary artificial light of 450 µmol quanta m-2 s-1 PAR (photosynthetically active radiation, 
=400–700 nm) at the plant level.
Determination of mineral content and osmolarity
Leaves were frozen at -20 °C and after thawing cell sap was obtained using a filter-paper disc-covered syringe. The osmolarity of the cell sap was measured with a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany). The cell sap was diluted with distilled water (1:40 to 1:100 depending on the Na+ and K+ concentrations) and boiled for 5 min. The filtered solution was used for the determination of Na+ and K+ concentrations using a flame photometer (Eppendorf, Netheler-Hinz, GmbH Hamburg, Germany).
Membrane vesicle isolation
Leaves were homogenized with a mixer in extraction medium containing 0.25–0.45 M mannitol depending on the actual osmotic potential of cell sap, 1 mM dithiothreitol (DTT), 2 mM EDTA, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), and 50 mM TRIS/MES (pH 7.8). The medium was always freshly prepared and 1 ml was used for 1 g of tissue. The homogenate was passed through four layers of cheesecloth and then centrifuged at 12000 g for 15 min. The supernatant was layered directly on a 25% (w/w) sucrose cushion containing 5 mM HEPES and 2 mM DTT, adjusted to pH 7.0 with TRIS, and centrifuged at 105000 g for 2 h (SW-28 rotor, Beckman, Munich, Germany). The interface between the supernatant and the 25% (w/w) sucrose cushion was carefully collected and diluted 3–4-fold with dilution buffer (1 mM DTT, 0.2 mM PMSF, 3 mM MgSO4, 50 mM HEPES, adjusted to pH 7.0 with KOH) and then centrifuged at 300000 g for 30 min (50.2Ti rotor, Beckman). The pellets were suspended in storage buffer (40% (v/v) glycerol, 1 mM DTT, 10 mM HEPES, adjusted to pH 7.0 with KOH). The suspension (membrane vesicle fraction containing tonoplast vesicles) was frozen in liquid nitrogen and stored at -75 °C until further use. All operations were carried out at 2–4 °C.
Various assays
Rates of ATP and PPi hydrolysis activity of membrane vesicles were calculated from the amount of inorganic phosphate released during an incubation period of 30 min at 37 °C at a protein concentration of 1 µg ml-1 in 0.5 ml assay medium. Inorganic phosphate was assayed using the method of Lin and Morales (Lin and Morales, 1977). For measurements of ATP-hydrolysis activity the assay medium contained 25 mM Tricine or MES, adjusted to pH 7.5 or 6.5, respectively, with TRIS, 3 mM Mg-ATP and 0.002% (w/v) of the non-ionic detergent Brij 58. V-ATPase activity was expressed as the difference between the values from measurements in the absence and the presence of 20 nM bafilomycin A1 at pH 7.5. Activity of F-ATPase was calculated from the difference of activities measured in the absence and presence of 1 mM sodium azide at pH 7.5. P-ATPase activity was determined at pH 6.5 as the activity difference of assays measured in the absence and in the presence of 0.1 mM sodium vanadate.
PPi hydrolysis activity was assayed in a reaction medium containing 25 mM Tricine, adjusted to pH 7.5 with TRIS, 0.002% (w/v) of the non-ionic detergent Brij 58, 3 mM MgSO4, 0.1 mM Na2MoO4, and 0.2 mM PPi. V-PPase activity was determined as the difference of activities measured in the presence or in the absence of 50 mM KCl (K+-stimulated PPase activity).
Protein concentrations were determined with Amino Black 10B following the procedure using bovine serum albumin as a protein standard (Popov et al., 1975).
SDS-PAGE and Western blot
SDS-PAGE was performed as described previously (Fischer-Schliebs et al., 1997) on slab gels containing 12.5% (w/v) acrylamide and by using the Laemmli buffer system (Laemmli, 1970). For Western blot analysis, proteins were transferred electrophoretically from acrylamide gels to Immobilon P® membranes (Millipore, Dassel, Germany) under conditions previously described (Fischer-Schliebs et al., 1997). After blocking free protein binding sites for 1 h in 1% (w/v) fat-free milk powder dissolved in phosphate buffered saline (PBS) the membranes were incubated with rabbit antisera directed against the V-PPase of Vigna radiata (anti-PPase; Maeshima and Yoshida, 1989), two different antisera against the V-ATPase holoenzyme of K. daigremontiana (ATP88a and ATP88b; for nomenclature see Fischer-Schliebs et al., 2000) or an antiserum directed against subunit A of the M. crystallinum V-ATPase (anti-A; Ratajczak et al., 1994), all diluted 1:1000 in PBS. An alkaline phosphatase-coupled goat-anti rabbit IgG (Western-Light®, Serva Tropix, Heidelberg, Germany; dilution 1:15000; incubation time 1 h) was used as a secondary antibody. Immunostaining was performed using nitroblue tetrazolium by the formation of an indigo dye-precipitate (Knecht and Dimond, 1983).
Immunoprecipitation
The V-ATPase holoenzyme was immunoprecipitated using Protein A-sepharose (Sigma, Deisenhofen, Germany) coupled antibodies directed against the V-ATPase subunit A of M. crystallinum (anti-A) as described previously (Fischer-Schliebs et al., 1997). Membrane vesicle preparations (100 µg protein) were solubilized for 30 min at 4 °C in 1% (w/v) Triton X-100 in PBS. Non-soluble material was removed by centrifugation at 12000 g for 20 min and the protein concentration of the supernatant (solubilized proteins) was determined. After immunoprecipitation of the V-ATPase protein from the supernatant by antibodies directed against V-ATPase subunit A of M. crystallinum coupled to Protein-A Sepharose (Sigma, Deisenhofen, Germany) protein determination of the supernatant was repeated. The amount of V-ATPase protein in the membrane vesicle fraction was calculated from the difference of protein concentration in the supernatant prior to and after immunoprecipitation.
Statistics
Identity of mean values was checked by t-test analysis after analysis of the identity of variances using the F-test performed with the program Costat (CoHort Software, Berkeley, California, USA).
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Results |
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Effects of NaCl, KCl and osmotic treatment on the growth of S. salsa
Irrigation of the plants with solutions containing different NaCl or KCl concentrations or 0.032 M PEG (iso-osmotic to 0.1 M NaCl solution) had effects on plant morphology. While plants treated with 0.1 and 0.4 M NaCl or 0.1 M KCl had thick stems, straight and succulent leaves and a high number of branches, controls and plants treated with 0.4 M KCl or PEG had thin, fragile, curled leaves and a low number of branches (data not shown). At the cellular level, light microscopical studies revealed that, independently of the treatment, cells contained large central vacuoles which occupied about 90% of the total cell volume during the whole time-course of the experiment (data not shown). Moreover, it has to be mentioned that the (i) lack of Kranz-type leaf anatomy (data not shown) and (ii) the
13C value of -32.5
(determined by Professor Dr H Rennenberg, Freiburg, Germany), which is typical for C3 plants (Ehleringer and Osmond, 1991), indicate that S. salsa is a C3 plant.
Compared to control plants treated with Hoagland solution the fresh weight of S. salsa shoots was significantly increased by 0.1 M NaCl, 0.4 M NaCl or 0.1 M KCl and significantly reduced by 0.032 M PEG (Fig. 1). The most prominent increase in fresh weight by a factor of 1.54 was observed after treatment of the plants with 0.4 M NaCl. Treatment with 0.1 M KCl led to a similar increase in fresh weight as treatment with 0.1 M NaCl. Interestingly, most of the plants irrigated with 0.4 M KCl wilted and died after 8 d of treatment. Thus, for plants treated with 0.4 M KCl only measurements of osmotic potential and ion content of the cell sap were performed. There was not sufficient material for growth analyses and the isolation of membrane vesicle fractions containing tonoplast vesicles required for proton pump analysis.
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Effects of NaCl, KCl and osmotic treatment on osmolarity and ion content of S. salsa leaves
Cell sap osmolarity increased with increasing NaCl or KCl concentration in the growth medium (Table 1). At a given salt concentration the cell sap osmolarity was higher in KCl-treated plants than in NaCl-treated plants. Cell sap Na+ concentration of plants treated with NaCl was markedly higher with increasing NaCl in the growth medium, but K+ concentration was lower. Similarly, cell sap K+ concentration of the plants treated with KCl was higher with increasing KCl in the growth medium, whereas Na+ concentration after 0.4 M KCl treatment was lower compared to controls. The cell sap osmolarity of 0.032 M PEG-treated plants was similar to that of 0.1 M NaCl-treated plants. PEG treatment led to an increase of cell sap Na+ concentration by a factor of 2.2 compared to controls, while cell sap K+ concentration was not significantly affected.
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Effects of NaCl, KCl and osmotic treatment on V-ATPase and V-PPase substrate hydrolysis activities
The purity of the isolated membrane vesicles was checked by characterizing the ATPase substrate hydrolysis activity in the absence and the presence of different specific inhibitors for membrane-bound H+-translocating ATPases. The pH optimum of V-ATPase activity of membrane vesicle fractions isolated from controls and plants treated with NaCl, KCl or PEG was at pH 7.5 (data not shown). In membrane vesicle fractions isolated from differentially treated plants during the whole time-course of the experiment, Bafilomycin A1-sensitive ATP-hydrolysis activity was 51.6±6.4% of total ATP-hydrolysis activity measured at pH 7.5. At the same pH value azide-sensitive ATP-hydrolysis activity was 10.1±6.5%. At pH 6.5, the pH-optimum of P-ATPase, 52.1±8.5% of total ATP-hydrolysis activity was sensitive to vanadate. This indicates that the membrane fractions analysed in this study contained tonoplast vesicles and that the contamination with inner mitochondrial membrane vesicles and plasmalemma vesicles was similar in all preparations.
While in controls V-ATPase substrate hydrolysis activity remained constant over the sampling period of 16 d, NaCl treatment of the plants led to a linear increase in V-ATPase activity (Fig. 2A). For the 0.1 M NaCl treatment and the 0.4 M NaCl treatment increases in V-ATPase activity of 0.747 µmol mg-1 tonoplast protein h-1 d-1 and 1.320 µmol mg-1 tonoplast protein h-1 d-1, respectively, have been measured (see slopes of linear regressions in Fig. 2A). Treatment of plants with 0.1 M KCl led to a transient increase in V-ATPase activity, on day 8 reaching a value which was not significantly different from the values obtained after 0.4 M NaCl (P=0.272) and 0.1 M NaCl (P=0.051) treatment. However, on day 16 of the experiment, ATP hydrolysis activity of membrane vesicle fractions isolated from 0.1 M KCl-treated plants dropped to a value similar to that obtained for controls (P=0.202). Membrane vesicle fractions from PEG-treated plants were isolated on day 8 of the experiment only. V-ATPase activity in these fractions was not significantly different from values obtained for controls (P=0.083).
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) Control; (
) 0.1 M NaCl; (•) 0.4 M NaCl; (
) 0.1 M KCl; (
) 0.032 M PEG. Solid straight lines plotted for data points obtained during analysis of membrane vesicle preparations isolated from controls, 0.1 M and 0.4 M NaCl-treated plants in (A) and controls and 0.1 M NaCl-treated plants in (B) are linear regressions with the following characteristics: control (A), y=-0.031x+24.89, r2=0.071; 0.1 M NaCl (A), y=0.747x+24.80, r2=0.920; 0.4 M NaCl (A), y=1.320x+25.35, r2=0.979; control (B), y=-0.014x+13.34, r2=0.059; 0.1 M NaCl (B), y=0.703x+13.53, r2=0.949. Dotted lines connecting data points indicate general trends.In controls V-PPase substrate hydrolysis activity (the difference of PPi hydrolysis activity measured in the presence and in the absence of 50 mM KCl) also remained constant during the time-course of the experiment (Fig. 2B
). However, the responses of V-PPase to different kinds of salt treatment were more complex compared to V-ATPase responses. Only in the case of 0.1 M NaCl treatment was a linear increase in V-PPase activity observed (0.703 µmol mg-1 tonoplast protein h-1 d-1). Treatment with 0.4 M NaCl and 0.1 M KCl led to a transient increase in V-PPase activity with a maximal activity on day 2 and day 8, respectively. At a significance level of 
=0.05 V-PPase activity of 0.4 M NaCl membrane vesicles was highest on day 2 and V-PPase activity of 0.1 M KCl membrane vesicles was highest on day 8 compared to all other fractions sampled on the respective days. On day 16 the activities of membrane fractions isolated from both 0.4 M NaCl and 0.1 M KCl were significantly lower compared to controls (P=0.019 and P=0.004, respectively). Data obtained measuring membrane vesicles isolated from PEG-treated plants indicate that osmotic stress markedly decreased V-PPase activity by a factor of 3.0 after 8 d of treatment.
Effects of NaCl, KCl and osmotic treatment on V-ATPase and V-PPase protein
In order to check whether the changes in V-ATPase activity due to different salt treatments and osmotic stress were correlated with changes in V-ATPase protein amount, quantitative immunoprecipitation of the V-ATPase holoenzyme from one selected membrane fraction per treatment isolated on day 8 of the experiment has been performed (Fig. 3). At a significance level of =0.005 V-ATPase amount related to total protein of membrane vesicle fractions isolated from plants treated with 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl was significantly higher compared to V-ATPase amount of control fractions. This is in good agreement with the V-ATPase activity data shown in Fig. 2A. However, in contrast to the activity data, V-ATPase amount in the fractions isolated from 0.1 M KCl-treated plants was higher compared to the amount present in the 0.4 M NaCl fraction. Moreover, V-ATPase amounts present in the 0.1 M NaCl and 0.4 M NaCl fractions were not significantly different, while V-ATPase activities of these two fractions were (compare Figs 2A, 3). These differences, however, should not be overinterpreted, since, as mentioned above, only one of three membrane fractions per treatment has been used for immunoprecipitation experiments. Moreover, due to its complicated experimental set-up, immunoprecipitation only leads to a relatively rough estimation of protein amount compared to the very precise data obtained by enzyme activity measurements. The ratio of V-ATPase activity to V-ATPase amount, which notably is independent of total tonoplast protein in the assays, was rather similar for controls and all salt treatments on day 8 (Table 3). By contrast to the different salt treatments, PEG treatment reduced the amount of the V-ATPase protein by a factor 1.45 compared to controls, and the V-ATPase activity/V-ATPase amount ratio was increased.
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To investigate the subunit composition of the S. salsa V-ATPase and putative changes in subunit composition due to different treatments, the cross-reaction of polypeptides present in membrane vesicle fractions isolated from S. salsa leaves after different treatments of plants were analysed using three different antisera directed against the V-ATPase of Kalanchoë daigremontiana (Fig. 4
). A summary of polypeptides cross-reacting with the different V-ATPase antisera and their apparent molecular masses is given in Table 2. The antisera ATP88a (Fig. 4A), ATP88b (Fig. 4B) and ATP95 (Fig. 4C) cross-reacted with 6, 7 and 5 polypeptides, respectively. In total, nine immunoreactive polypeptides could be distinguished. Polypeptides exhibiting molecular masses of 66, 55, 36, and 18 kDa were recognized by all three antisera, while polypeptides 52, 48, 35, 29, and 16 kDa were only recognized by one or two forms. The polypeptide pattern was identical in membrane fractions isolated from leaves of controls and plants grown with NaCl, KCl and PEG. The antiserum anti-Di directed against the M. crystallinum Di polypeptide (32 kDa) immunostained four polypeptides with apparent molecular masses of 66, 48, 36, and 20 kDa (Fig. 4D). While the three high molecular mass polypeptides were visible in all fractions, the 20 kDa polypeptide was absent in controls.
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Due to its lower total protein amount compared to the V-ATPase, the V-PPase protein amount was not studied by immunoprecipitation experiments. Thus, no data can be presented for the correlation of V-PPase activity changes and V-PPase protein amount. On the other hand, Western-blot analyses revealed that a 69 kDa polypeptide was immunostained by an antiserum directed against the V. radiata V-PPase in all fractions (Fig. 5
). Interestingly, in membrane fractions isolated from PEG-treated plants on day 8 of the experiment, an additional polypeptide exhibiting an apparent molecular mass of 54 kDa cross-reacted with the antiserum.
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Discussion |
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Considering shoot fresh weight as an indicator of plant growth capacity, it is obvious from the results obtained in the present study that S. salsa is a typical halophyte growing optimally when irrigated with a solution containing 0.4 M NaCl (Fig. 1
). The fact that growth of plants irrigated with 0.1 M NaCl or 0.1 M KCl was similar, and for both was significantly higher compared to controls, indicates that growth stimulation is not specific for Na+. However, it has to be mentioned that this is only true to a certain extent, since plants grown at 0.4 M KCl wilted and died soon after the onset of treatment. Thus, S. salsa is especially Na+-tolerant and to a lower extent K+ tolerant. For the C4-photosynthesis species of Suaeda, namely S. aegyptiaca and S. monoica, a growth-stimulating effect of Na+ has already been reported (Eshel, 1985a, b). However, in the same studies neither growth stimulation nor growth inhibition caused by K+ (up to concentrations of 125 or 150 mM KCl) has been observed. Thus, with respect to K+ responses S. salsa seems to be unique in the genus Suaeda as far as is known. Whether this is due to the different mode of photosynthesis of S. salsa (C3-photosynthesis) compared to the other Suaeda species (C4-photosynthesis) remains an open question.
K+ is the most abundant cation in higher plants, and it is crucial for plant nutrition, growth, tropism, enzyme activities, ion homeostasis, and osmoregulation (Epstein, 1972; Zhu et al., 1998). K+ has also been considered to play a role in salinity toxicity in general (Läuchli and Hepler, 1990; Niu et al., 1995) and especially in non-halophytes (Zhu et al., 1998). An Na+-induced decrease of tissue K+ content has been considered as a contributor to salinity toxicity (Ben-Hayyim et al., 1987; Nakamura et al., 1990). However, K+ nutrition does not seem to be a critical factor in salt tolerance of S. salsa because growth was promoted although K+ concentration in the cell sap was markedly decreased by NaCl treatment.
K+ is a main cation of the cytoplasm. The optimal cytoplasmic K+ concentration ranges between 50 and 250 mM (Niu et al., 1995). The K+ concentration in the leaf cell sap of the well growing S. salsa plants exposed to 0.1 M KCl for 8 d was about 0.3 M. Taking into account that in S. salsa leaf cells the vacuole occupies more than 90% of the total cell volume, it is obvious that the bulk of the K+ ions must be stored in the vacuole. The leaf cell sap K+ concentration of plants exposed to 0.4 M KCl reached about 1.28 M. In this case, the plants showed significant symptoms of injury as described above. The mechanism of injury by high K+ is not known. For other Suaeda species it was found that KCl treatment led to a poorer water use efficiency than NaCl treatment (Eshel, 1985b), suggesting that high K+ concentrations disturb photosynthesis and transpiration. From electrophysiological studies on Hordeum vulgare cells, it was suggested that K+ uptake into the vacuole, except under conditions of severe K+ deficiency is active and might be mediated either by direct V-PPase K+ transport or by a 1:1 H+:K+ symport (Walker et al., 1996). Thus, highly toxic K+ concentrations in the cytoplasm might occur when these active transport mechanisms reach the thermodynamic limits. The compartmentation of Na+ in the vacuole seems to be more efficient than the compartmentation of K+, since a high Na+ concentration of 1.02 M in the cell sap (a concentration which was not significantly different from the cell sap K+ concentration of 1.28 M measured after treatment with 0.4 M KCl [see Table 1]; P=0.097) led to optimal growth rather than growth inhibition.
The transport of both K+ (putatively via a vacuolar cation channel) or Na+ (via an Na+/H+ antiporter; Barkla and Pantoja, 1996), has to be energized by the action of primary-active proton pumps at the tonoplast, i.e. the V-ATPase and/or the V-PPase. Thus, the effects of different salt treatments and osmotic stress on the activity of vacuolar proton pumps were studied. It turned out that NaCl at both concentrations used increased the activity of the V-ATPase during the time-course of the experiment (Fig. 2A). An increase in V-ATPase activity under salinity seems to be a general response of salt-tolerant plants (Binzel and Ratajczak, 2001, and references therein). As shown by immunoprecipitation experiments in S. salsa this increase in activity was achieved by an increase in V-ATPase protein amount (Fig. 3). In plants treated with 0.1 M KCl, only a transient increase in V-ATPase activity was observed which might be explained by the requirement of higher tonoplast energization during the early phase of KCl treatment. Under all of these three salt treatments the activity/amount ratio of the V-ATPase remained similar to the control (Table 3). The fact that under osmotic stress V-ATPase activity was not significantly different compared to controls indicates that mechanisms sensing ion concentrations seem to be involved in the up-regulation of V-ATPase protein amount.
No clear pattern was detected for NaCl-dependent V-PPase activity changes (Fig. 2B). While treatment with 0.1 M NaCl increased V-PPase activity, treatment with 0.4 M NaCl led to a small transient increase 2 d after the onset of salt treatment. A transient increase of V-PPase activity was also observed during treatment with 0.1 M KCl, however, in this case peak V-PPase activity was measured on day 8 of the experiment. Interestingly, neither treatment with NaCl or KCl led to a decrease in V-PPase activity to values dramatically lower compared to controls, as it was found to occur in the C3/CAM-intermediate halophyte M. crystallinum under NaCl salinity (Bremberger et al., 1988; Bremberger and Lüttge, 1992a; Rockel et al., 1994). In this respect, it has to be mentioned that in M. crystallinum a decrease in V-PPase was also observed when plants reached the CAM state of photosynthesis in the absence of NaCl as the result of a developmental programme (Rockel et al., 1994). Thus, in M. crystallinum the decrease in V-PPase activity might be correlated to CAM expression and not to NaCl salinity per se. In cultured cells of D. carota (Colombo and Cerana, 1993) and Acer pseudoplatanus (Zingarelli et al., 1994), V-PPase activity was induced by NaCl salinity. On the other hand, NaCl-treatment of the halophyte S. maritima (Leach et al., 1990) and cultured cells of Nicotiana tabacum (Reuveni et al., 1990), did not change V-PPase activity, while it was decreased after salt treatment in root cells of Vigna radiata (Nakamura et al., 1992). This clearly shows that NaCl salinity responses of the V-PPase are dependent on the plant species and cannot be generalized.
It is still an open question, why the tonoplast contains two primary-active proton pumps, i.e. the V-ATPase and the V-PPase. Thus, a lot of work has been done in the last few years in order to find physiological conditions which would require greater activity of the V-PPase than of the V-ATPase. However, changes of a variety of growth conditions in different experimental plants led to a variety of responses of V-ATPase and V-PPase and no clear correlative pattern of activation or deactivation of both proton pumps has been found to date (Fischer-Schliebs et al., 1998, and references therein). In the present study, only in the case of 0.1 M NaCl treatment was V-PPase activity markedly increased over the entire duration of the experiment. However, in this case V-ATPase activity was also increased in a similar way. All other treatments only led to a small transient increase of V-PPase activity or to a decrease of activity compared to controls. Thus, under salt stress and osmotic stress conditions in S. salsa, V-PPase activity seems to be less important physiologically than V-ATPase activity.
Three different antisera directed against the K. daigremontiana V-ATPase holoenzyme have been used to determine the S. salsa V-ATPase subunit composition. Four out of a total of nine polypeptides were immunostained by all three antisera used (Table 2), i.e. polypeptides exhibiting molecular masses of 66, 55, 36, and 18 kDa, which most probably represent V-ATPase subunits A, B, D, and c, respectively, according to comparison with the apparent molecular masses of V-ATPase subunits of different species (Ratajczak, 2000, and references therein). The other polypeptides, which were immunostained by only one or two of the antisera used (52, 48, 35, 29, and 16 kDa) might represent other V-ATPase subunits located either in the central or the peripheral stalk or in the Vo domain of the holoenzyme. The number of nine polypeptides is very close to the number of 12 subunits present in the yeast V-ATPase holoenzyme (Stevens and Forgac, 1997), and one might suspect that the three missing V-ATPase subunits might not cross-react with the antisera used in this study.
Looking at the immunostaining results obtained with the three V-ATPase antisera used, there was no difference in S. salsa V-ATPase subunit composition depending upon the different treatments of the plants. However, immunostaining using an antiserum against the M. crystallinum polypeptide Di showed that a 20 kDa polypeptide was present in fractions isolated from NaCl-, KCl- and PEG-treated plants, which was absent in controls. The polypeptide Di was shown to represent the C-terminus of subunit B of the M. crystallinum V-ATPase occurring in vivo after the shift from C3-photosynthesis to CAM (Bremberger et al., 1988; Bremberger and Lüttge, 1992b; Zhigang et al., 1996). In a recent study it was shown that the formation of Di in vitro is mediated by a specific protease and/or reactive oxygen species (Krisch et al., 2000), indicating that the processing of subunit B is more related to the occurrence of oxidative stress than to the shift of photosynthetic metabolism. Thus, it might be suspected, that the S. salsa 20 kDa polypeptide found under salt and drought conditions is a fragment of subunit B produced under conditions of elevated oxidative stress as it is often associated with salinity (Dat et al., 2000). The cross-reaction of the antiserum directed against the polypeptide Di with subunits A, B and D had already been observed in Western blots of tonoplast proteins isolated from M. crystallinum (Zhigang et al., 1996) and can be explained as follows. Since Di is a fragment of subunit B, it is obvious that subunit B is decorated by the antiserum. The cross-reaction with subunit A might be due to the presence of immunogenic splitting products of subunit A contaminating the Di antigen (Zhigang et al., 1996). Finally, cross-reaction with subunit D might be a result of Di antigen preparation. Prior to immunization of rabbits, the Di polypeptide was separated from other proteins by denaturing gel electrophoresis and blotted onto nitrocellulose membranes. After protein staining, the band corresponding to Di was excised from the nitrocellulose membrane (Zhigang et al., 1996). Due to the similar molecular mass of subunit D and the Di polypeptide, it cannot be excluded that small amounts of subunit D contaminated the Di antigen.
A polypeptide exhibiting an apparent molecular mass of 69 kDa was immunostained by an antiserum against the V. radiata V-PPase. From molecular mass comparison with V-PPases from other species (for reviews see Leigh et al., 1994; Zhen et al., 1997), this polypeptide most probably represents the S. salsa V-PPase. Interestingly, PEG treatment led to the immunostaining of an additional 54 kDa polypeptide cross-reacting with the antiserum, which was not present in controls and only slightly visible in preparations from plants treated with 0.1 M KCl. This polypeptide might represent a proteolytic fragment of the V-PPase occurring due to the change in growth conditions, suggesting that under stress conditions V-PPase protein is degraded.
In conclusion, the data presented show that S. salsa is a typical halophyte exhibiting a high degree of salt tolerance. On the other hand, the plant is sensitive to osmotic stress and high concentrations of potassium. The main strategy of salt-tolerance seems to be a quantitative up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, via an increased amount of the enzyme without any qualitative structural changes. Conversely, the V-PPase only seems to play a minor role.
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Acknowledgments |
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We are grateful for financial support from the NSFC (National Natural Science Research Foundation of China project No. 30070069), the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 199, projects B2 and B3) and Deutscher Akademischer Austauschdienst (DAAD; stipend for a research stay of BW in Darmstadt). We thank Professor Dr Masayoshi Maeshima (Nagoya, Japan) for generously providing an antiserum directed against the Vigna radiata V-PPase. We also thank Professor Dr Heinz Rennenberg (University of Freiburg, Germany) for carbon isotope analysis.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 6151 164630. E-mail: luettge{at}bio.tu\|[hyphen]\|darmstadt.de1
2 Permanent address: Department of Biology, Shandong Normal University, Jinan 250014, PR China.
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Abbreviations |
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ATP, adenosine triphosphate; CAM, crassulacean acid metabolism; DTT, dithiothreitol; EGTA, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid; HEPES, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulphonic acid); MES, 2-morpholinoethanesulphonic acid; P-ATPase, plasmalemma H+-translocating adenosine triphosphatase; PBS, phosphate buffered saline; PMSF, phenylmethylsulphonyl fluoride, PPi, inorganic pyrophosphate; PVP, polyvinylpyrrolidone; Tricine, N-tris-(hydroxymethyl)-methylglycine; V-ATPase, vacuolar-type H+-translocating adenosine triphosphatase; V-PPase, vacuolar-type H+-translocating inorganic pyrophosphatase; PEG, polyethylene glycol.
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