This Article Abstract Full Text (PDF) All Versions of this Article: 134/1/452    most recent pp.103.030361v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Rubio, L. Articles by Botella, M. A. Search for Related Content PubMed PubMed Citation Articles by Rubio, L. Articles by Botella, M. A. Agricola Articles by Rubio, L. Articles by Botella, M. A.

ENVIRONMENTAL STRESS AND ADAPTATION

Regulation of K⁺ Transport in Tomato Roots by the TSS1 Locus. Implications in Salt Tolerance¹

Departamento de Biología Vegetal (L.R., A.L.-R., M.J.G.-S., J.A.F.) and Departamento de Biología Molecular y Bioquímica (A.R., V.V., M.A.B.), Universidad de Málaga, 29071 Málaga, Spain; and Departamento de Biología Vegetal, Laboratorio de Bioquímica, Facultad de Agronomía, Avda Garzon 780, 12900 Montevideo, Uruguay (O.B.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K⁺ and hypersensitivity to Na⁺ and Li⁺. Increased Ca²⁺ in the culture medium suppressed the Na⁺ hypersensitivity and the growth defect on low K⁺ medium of tss1 seedlings. Interestingly, removing NH₄⁺ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH₄⁺-insensitive component of K⁺ transport. We performed electrophysiological studies to understand the contribution of the NH₄⁺-sensitive and -insensitive components of K⁺ transport in wild-type and tss1 roots. Although at 1 mM Ca²⁺ we found no differences in affinity for K⁺ uptake between wild type and tss1 in the absence of NH₄⁺, the maximum depolarization value was about one-half in tss1, suggesting that a set of K⁺ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca²⁺ concentration. In the presence of NH₄⁺, a reduced affinity for K⁺ was observed in both types of seedlings, but tss1 at 1 mM Ca²⁺ exhibited a 2-fold higher K_m than wild type did. This defect was again corrected by raising the external concentration of Ca²⁺. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH₄⁺-sensitive and -insensitive components of K⁺ transport at low Ca²⁺ concentrations and that this defective transport is rescued by increasing the concentration of Ca²⁺. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca²⁺ involved in K⁺ nutrition and salt tolerance.

K⁺ uptake occurs through different transport mechanisms depending on the external K⁺ concentration. Carriers that exhibit a high affinity for K⁺ and some K⁺ inward-rectifying (KIR) channels work at low K⁺ (<1 mM K⁺), whereas different types of channels, such as KIR channels or nonselective cation channels (NSCCs), are involved when external K⁺ concentration is in the millimolar range (Rodriguez-Navarro, 2000; Schachtman, 2000; Demidchik et al., 2002; Véry and Sentenac, 2003). Although some KIR channels (AKT type) may function at low K⁺ (Hirsch et al., 1998), carriers are thought to be responsible for the majority of K⁺ uptake in the micromolar range (Maathuis and Sanders, 1997; Rubio et al., 2000). As previously reported in Arabidopsis (Spalding et al., 1999), in tomato (Lycopersicon esculentum) there appear to be two distinct mechanisms involved in K⁺ uptake (Borsani et al., 2001). The first mechanism is insensitive to inhibition by NH₄⁺ and, by analogy to Arabidopsis, may correspond to inward-rectifying K⁺ channels such as the AKT1 channel (Spalding et al., 1999). The second mechanism is inhibited by NH₄⁺ and may correspond to an active K⁺ transport coupled to the H⁺ electrochemical potential gradient (Santa-María et al., 2000). In addition to its effect on K⁺ uptake, NH₄⁺ can result toxic for most plant species, including tomato. Different explanations for this toxicity besides K⁺ transport inhibition have been suggested. These include intracellular pH disturbance, carbon deprivation, and ionic imbalance associated with a decrease in the internal concentration of K⁺, Mg²⁺, and Ca²⁺ (Britto et al., 2001; Kronzucker et al., 2001).

Ca²⁺ plays multiple roles in root hair development, ionic homeostasis, and stress-induced responses (Sanders et al., 1999; Reddy, 2001). A rise in the external Ca²⁺ concentration can improve the K⁺/Na⁺ selectivity (Hasegawa et al., 2000), increasing the K⁺ uptake under salt stress. An inhibitory effect on the activity of KIR channels by Ca²⁺ has been reported (White, 1997). In addition, different responses of NSCCs to Ca²⁺ such as activation (Ca²⁺-activated NSCCs), partial inhibition (voltage-insensitive NSCCs), and increased permeability (hyperpolarization-activated NSCCs) have been shown (Demidchik et al., 2002). Ca²⁺-mediated responses involve temporal and spatial oscillations in cytosolic free Ca²⁺ activity (Evans et al., 2001). For this reason, the free Ca²⁺ concentration in the cytosol, usually in the nanomolar range, is strongly regulated. Interaction between Ca²⁺ concentrations, K⁺ transport, and salinity has been reported in Arabidopsis (Liu and Zhu, 1998; Hasegawa et al., 2000; Zhu, 2002). A myristoylated Ca²⁺-binding protein encoded by SOS3 presumably senses the salt-elicited calcium signal and translates it to downstream responses (Liu and Zhu, 1998; Ishitani et al., 2000). SOS3 interacts with and activates SOS2, a Ser/Thr protein kinase (Halfter et al., 2000; Liu et al., 2000). SOS2 and SOS3 regulate the expression level of SOS1, a salt tolerance effector gene encoding a plasma membrane Na⁺/H⁺ antiporter (Shi et al., 2000). More importantly, SOS2 and SOS3 are required for the activation of SOS1 transport activity (Qiu et al., 2002; Quintero et al., 2002).

We have isolated tomato mutants in the search for salt and osmotic hypersensitive mutants using a molecular genetic approach (Borsani et al., 2001, 2002). The tss1 mutant behaves as a phenocopy of the Arabidopsis sos3 mutant because it is hypersensitive to Na⁺ and exhibits a growth defect on low K⁺ (Borsani et al., 2001). K⁺-dependent membrane potential depolarizations indicated impaired K⁺ uptake in tss1. As occurs is sos3, increased Ca²⁺ concentration in the culture medium can partially suppress the Na⁺ hypersensitivity of tss1 seedlings and completely suppress the growth defect on low K⁺ medium (Borsani et al., 2001). These phenotypes suggest that, as SOS3, TSS1 is required to amplify a Ca²⁺ signal during Na⁺ stress and K⁺ starvation.

In this work, we show that the hypersensitivity to Na⁺ and Li⁺ and the reduced growth in low K⁺ of tss1 is suppressed when the growth medium does not contain NH₄⁺. Interestingly, this NH₄⁺-dependent phenotype was not mimicked by the Arabidopsis sos3 mutant. The aim of this work is to study the effect of Ca²⁺ and the influence of NH₄⁺ on both root growth and K⁺ transport in wild-type and tss1 root seedlings, with special emphasis on K⁺ transport kinetics.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Isolation of New Mutants at the TSS1 Locus

A previous screening of 600 M₂ seed families resulted in the isolation of three tomato mutants (tss1-1, tss1-2, and tss2) defining two genetic loci required for NaCl tolerance (Borsani et al., 2001). A further 2,000 ethylmethane sulfonate M₂ seed families were screened looking for hypersensitivity to NaCl. The screening resulted in the isolation of three additional mutants hypersensitive to NaCl. As shown in Table I, analysis of the mutant segregation within each M₂ family showed an approximately 3:1 segregation ratio of wild type:mutant (Table I). Thus, all new tss mutants were caused by single recessive nuclear mutations. Complementation analyses indicated that the three mutants were allelic to tss1-1 (data not shown). We determined the degree of NaCl hypersensitivity from the five tss1 alleles. Table I shows the concentrations of NaCl at which the root elongation rate decreased by 50% relative to medium without salt (I₅₀). tss1-1, tss1-2, and tss1-3 showed similar I₅₀ values (68, 70, and 67 mM NaCl, respectively). tss1-4 and tss1-5 were weaker alleles with I₅₀ values of 125 and 115 mM NaCl, respectively. All subsequent physiological studies were performed using tss1-3 (hereafter referred to as tss1).

Hypersensitivity of tss1 Seedlings to Na⁺, Li⁺, and the Growth Defect on Low K⁺ Is Abolished in the Absence of NH₄⁺

tss1 mutants were isolated based on their hypersensitivity to grow on Murashige and Skoog medium containing 125 mM NaCl (Borsani et al., 2001, this work). tss1 was hypersensitive to all NaCl concentrations analyzed (Borsani et al., 2001). We also determined that tss1 was affected in K⁺ uptake, which in turn would render the plant hypersensitive to NaCl. Surprisingly, we found that tss1 was not hypersensitive to NaCl when a new growth medium was employed (see "Materials and Methods"). Several differences in salt concentrations are found between these two media. However, the main qualitative difference found is the absence of NH₄⁺ in the new medium, whereas the concentration of NH₄⁺ in the Murashige and Skoog medium is 20 mM. The inhibitory effects of NH₄⁺ on K⁺ uptake have been reported previously (Scherer et al., 1984; Vale et al., 1987; Spalding et al., 1999; Santa-María et al., 2000). Supplementing the medium with 5 mM NH₄⁺ restored the tss1 hypersensitivity to NaCl (Fig. 1), proving that the abolishment of NaCl hypersensitivity in the new medium was due solely to the lack of NH₄⁺.

View larger version (107K): [in this window] [in a new window]   Figure 1. tss1 is only hypersensitive to Na+ only when the medium contains NH4+. Wild type (WT) and tss1 3-d-old seedlings with 1.5-cm-long roots grown on vertical agar plates on basal medium were transferred to new medium supplemented with 125 mM NaCl in the absence (-NH4+) or presence of 5 mM NH4+ (+NH4+) and allowed to grow for another 2 d.

Next, we investigated whether tss1 hypersensitivity was abolished in media lacking NH₄⁺ at different concentrations of NaCl. As shown in Figure 2A, no differences between wild-type and tss1 seedlings were observed when the media did not contain NH₄⁺, whereas tss1 seedlings remained hypersensitive in NH₄⁺-containing media. We had shown previously that tss1 was hypersensitive to Li⁺ (Borsani et al., 2001). Li⁺ is considered a more toxic analog of Na⁺ and has been used to mimic Na⁺ toxicity without the osmotic stress component associated with NaCl. We also determined that Li⁺ hypersensitivity of tss1 was dependent on the presence of NH₄⁺ in the growth medium (Fig. 2B). tss1 growth was impaired at low K⁺ concentrations (<1 mM K⁺), suggesting that tss1 is most likely affected in an NH₄⁺-insensitive K⁺ transport mechanism (Borsani et al., 2001). When we analyzed the growth defect of tss1 on low K⁺ in the presence or absence of NH₄⁺, reduced growth was observed only in media with NH₄⁺. Removal of NH₄⁺ also stimulated root growth of wild-type plants at increasing K⁺ concentrations (Fig. 2C).

View larger version (20K): [in this window] [in a new window]   Figure 2. Removing NH4+ from the growth medium suppresses the hypersensitivity of tss1 to Na+ and Li+ and the growth defect of tss1 at low K+. Wild-type (solid line) and tss1 (dashed line) seedlings were grown on basal medium for 2 d. The seedlings were then transferred for 3 d to new medium supplemented with different external concentrations of NaCl (A) and LiCl (B), in the absence (white circles) or presence (solid circles) of 5 mM NH4. C, Wild-type (solid line) and tss1 (dashed line) seedlings were germinated and grown for 3 d on K+- and Ca2+-free medium. Then, seedlings were transferred to new K+- and Ca2+-free medium supplemented with the indicated concentrations of KCl. Root growth in the first 2 d was not measured so that the effect of residual K+ carried over from the germination medium was minimized. Root elongation was measured 3 d later. Error bars = SD (n = 10).

sos3 Hypersensitivity to Na⁺ and Li⁺ Is Independent of NH₄⁺

tss1 mutants resemble the sos3 mutant isolated previously in Arabidopsis (Liu and Zhu, 1997, 1998). Both sos3 and tss1 plants exhibited normal growth except when challenged with low K⁺ or salt stress. As in sos3, increasing external Ca²⁺ suppressed the growth defect on low K⁺ medium and partially suppressed the salt hypersensitivity phenotype of tss1. Therefore, we analyzed whether sos3 hypersensitivity to NaCl and LiCl was abolished in the absence of NH₄⁺ as occurred with tss1. As shown in Figure 3A, the sos3 mutant remained hypersensitive to NaCl whether or not the media contained NH₄⁺. Moreover, the degree of NaCl hypersensitivity of sos3 was similar in both media (Fig. 3A). It is interesting to note that Arabidopsis wild-type seedlings markedly improved their NaCl tolerance when the NH₄⁺ was removed from the growth media (Fig. 3A). The improved NaCl tolerance of wild-type Arabidopsis seedlings without NH₄⁺ was not observed in wild-type tomato (Fig. 2A). As for NaCl, sos3 remained hypersensitive to LiCl in NH₄⁺-free media. However, in contrast to Na⁺, the addition of NH₄⁺ to the growth media had a beneficial effect (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Figure 3. Hypersensitivity to Na+ and Li+ of sos3 is independent of the presence of NH4+ in the growth medium. Arabidopsis seedlings were grown on basal medium for 4 d and then transferred for 7 additional d to new medium supplemented with different external concentrations of NaCl (A) and LiCl (B), in the absence (white squares) or presence (solid squares) of 5 mM NH4+. Solid line, Wild type; dashed line, sos3. Error bars = SD (n = 10).

Effect of Ca²⁺ and NH₄⁺ on K⁺ Uptake

Our previous results suggest that the reduced growth on low K⁺ medium and the hypersensitivity to both Na⁺ and Li⁺ could be due to a defective K⁺ uptake of tss1 (Borsani et al., 2001). Because these defects are abolished when the media lacks NH₄⁺, we speculate that NH₄⁺ is inhibiting a K⁺ uptake system that become functional after NH₄⁺ is removed from the media. Previous studies using the Arabidopsis akt1 mutant indicated that at low K⁺ concentrations (10-100 µM K⁺), K⁺ uptake is dependent on two classes of transport mechanisms operating in parallel (Hirsch et al., 1998; Spalding et al., 1999). The first mechanism is uninhibited by NH₄⁺ and corresponds to the inward-rectifying K⁺ channel AKT1 (Sentenac et al., 1992; Spalding et al., 1999). The second mechanism is NH₄⁺ sensitive and corresponds to non-AKT1 transporters (Spalding et al., 1999). There is evidence indicating that HAK transporters may be involved in this NH₄⁺-sensitive pathway (Santa-María et al., 2000). The reduced growth of tss1 is only observed at concentrations of Ca²⁺ ranging from 0.15 to 1 mM. Increasing the external Ca²⁺ over this value completely suppressed the growth defect of tss1 at low K⁺ (Borsani et al., 2001). Therefore, we have studied the effect of two different external Ca²⁺ concentrations, 1 and 5 mM, in the transport kinetics of K⁺ in wild-type and tss1 root seedlings in the absence or presence of NH₄⁺.

Electrophysiological experiments revealed similar resting membrane potentials (E_m) at 1 and 5 mM external Ca²⁺ concentrations both in wild-type and tss1 root cells either in the absence (-160 ± 12 mV) or presence (-153 ± 12 mV) of 1 mM NH₄⁺ (ANOVA, = 0.01). In medium without NH₄⁺, increasing K⁺ concentrations (from 0.1-1000 µM KCl) elicited rapid membrane depolarizations in both wild-type and tss1 seedlings grown at 1 or 5 mM external Ca²⁺ concentrations (Fig. 4, A and B). The membrane depolarization values showed a saturation kinetics model and were fitted to the Michaelis-Menten equation (Table II). Wild-type seedlings exhibited similar K_m values whether the media contained 1 or 5 mM external Ca²⁺, although a slight increase in D_max value was reported in seedlings adapted to 5 mM external Ca²⁺ (Table II). Root cells of tss1 seedlings grown at 1 mM external Ca²⁺, showed a similar K_m value but approximately 50% of the D_max value obtained for the wild type in similar conditions (Table II). However, when wild-type and tss1 seedlings were grown at 5 mM external Ca²⁺, no differences were observed in kinetics parameters, indicating that K⁺ transport was re-established (two way ANOVA, = 0.01).

View larger version (17K): [in this window] [in a new window]   Figure 4. Membrane potential depolarizations induced by increasing K+ concentrations in wild-type (solid line) and tss1 (dashed line) root cells. Seeds were germinated in distilled water and grown for 6 d in the absence of NH4+ at 1 mM NaCl and at 1 (A) or 5 (B) mM CaCl2. Values showed a saturation kinetics model and were fitted to the Michaelis-Menten equation. The calculated kinetics parameters are shown in Table II. Data are means ± SD (n = 5).

Evidence for an inhibitory effect of NH₄⁺ on K⁺ transport has been reported in other plant species on the basis of short-term radiometric studies (Scherer et al., 1984; Vale et al., 1987, 1988; Wang et al., 1996; Spalding et al., 1999; Santa-María et al., 2000). In medium containing NH₄⁺, increasing K⁺ concentrations also evoked rapid membrane depolarizations in both wild-type and tss1 seedlings (Fig. 5, A and B). As before, the membrane depolarization values showed a saturation kinetics model that could be fitted to the Michaelis-Menten equation. However, the K⁺ concentration needed to reach saturation was around 10 mM. When kinetics parameters for K⁺ uptake in the presence of 1 mM NH₄⁺ were calculated, clear differences between wild type and tss1 were observed (Table II). In wild-type and tss1 seedlings, the K_m value for K⁺ increased at least 100-fold, regardless of the external Ca²⁺ concentration, indicating that the affinity for K⁺ uptake is greatly dependent on the presence of NH₄⁺ (Table II). At 1 mM Ca²⁺, tss1 root cells exhibited a higher K_m and a lower D_max value than wild type (Fig. 5A; Table II). However, the addition of 5 mM Ca²⁺ to the medium suppressed the kinetics differences between wild-type and tss1 seedlings (Fig. 5B; two-way ANOVA, = 0.01).

View larger version (17K): [in this window] [in a new window]   Figure 5. Membrane potential depolarizations induced by increasing K+ concentrations in wild-type (solid line) and tss1 (dashed line) root cells. Seeds were germinated in distilled water and grown for 6 d in the presence of 1 mM NH4Cl at 1 mM NaCl and at 1 (A) or 5 (B) mM CaCl2. Values showed a saturation kinetics model and were fitted to the Michaelis-Menten equation. The calculated kinetics parameters are shown in Table II. Data are means ± SD (n = 5).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Identification of New tss1 Alleles

In plants, K⁺ plays an essential role as an osmoticum and charge carrier (Rodriguez-Navarro, 2000). The capacity of plants to maintain a high cytosolic K⁺ to Na⁺ ratio is likely to be one of the key determinants of plant salt tolerance (Maathuis and Amtmann, 1999). The tss1 mutant is specifically hypersensitive to growth inhibition by Na⁺ or Li⁺ and has impaired growth at low K⁺ (<1 mM K⁺) when external Ca²⁺ concentration is low. We have shown that all phenotypes cosegregated, indicating that mutations in TSS1 were responsible for all three phenotypes (Borsani et al., 2001). Five mutants have been identified in the TSS1 locus and three of them, tss1-1, tss1-2, and tss1-3, appeared very similar in their sensitivity to NaCl (Table I), which suggests that they are null mutants.

Root Growth of tss1 in Low K⁺ or High Na⁺ Is Dependent on Ca²⁺ and NH₄⁺

Based on previous data (Borsani et al., 2001), tomato seems to have two distinct mechanisms for K⁺ uptake similar to those reported previously in Arabidopsis and barley (Hordeum vulgare; Spalding et al., 1999; Santa-María et al., 2000). The first one is a mechanism insensitive to inhibition by NH₄⁺ that, by analogy to Arabidopsis, may correspond to inward-rectifying K⁺ channels such as the AKT1 channel. The second is a mechanism inhibited by NH₄⁺, which may correspond to an active K⁺ transport coupled to the H⁺ electrochemical gradient. Based on previous data (Borsani et al., 2001), we can speculate that the NH₄⁺-sensitive component of tss1 is functional (at least partially) because this reduced growth of tss1 in low K⁺ and low Ca²⁺ is reverted in media lacking NH₄⁺. However, removing NH₄⁺ from the growth media not only suppressed the growth defect at low K⁺ but also the tss1 hypersensitivity to Na⁺ and Li⁺ (Fig. 2, A-C). These results suggest that the hypersensitivity to Na⁺ and Li⁺ of tss1 can be due to a defective in K⁺ uptake in the presence of these ions that is rectified when the NH₄⁺-sensitive component becomes active.

tss1 tomato mutants resemble the Arabidopsis sos3 mutant. This prompted us to speculate that TSS1 could be a SOS3 ortholog in tomato, therefore encoding some type of Ca²⁺ sensor (Liu and Zhu, 1997, 1998). However, the hypersensitivity to Na⁺ and Li⁺ of sos3 was not overcome by removing NH₄⁺ from the growth media (Fig. 3). It is possible that TSS1 and SOS3 are not ortholog genes and, despite similarities in phenotype, they regulate different targets. Alternatively, Arabidopsis may lack or not express the genes encoding the NH₄⁺-sensitive K⁺ transporters required for growth at low K⁺ or high salt. It is known that SOS3, a Ca²⁺ sensor, together with SOS2, encoding a protein kinase, regulates SOS1, an Na⁺/H⁺ antiporter (Zhu, 2002). Therefore, the Na⁺ hypersensitivity of sos3 also could be explained by factors other than K⁺ nutrition. Despite the advantages of Arabidopsis as a model plant, it is not clear if it can be used as a universal system to understand the physiology of K⁺ uptake or Na⁺ tolerance. As a consequence, additional studies in other plants might be necessary to understand the whole picture of K⁺ transport and Na⁺ tolerance.

K⁺ Uptake Mechanisms Affected in tss1

Our results revealed that tomato takes up K⁺ from low micromolar K⁺ concentrations when the plants are grown in conditions of K⁺ deficiency (Table II). Because relatively similar kinetics parameters for K⁺ uptake were found in wild-type seedlings in media lacking NH₄⁺ at 1 mM Ca²⁺ and 5 mM Ca²⁺, we can speculate that the same type of K⁺ transporters are active regardless of external Ca²⁺ concentration. However, at 1 mM Ca²⁺, tss1 exhibits a defective K⁺ uptake (Fig. 4A). In these conditions, root cells of both tss1 and wild type showed a similar K_m value, but tss1 exhibited a lower D_max, an estimation of K⁺ transport V_max (Fig. 4A; Table II). This may be interpreted as a decrease in the number of active K⁺ transporters (Malhotra and Glass, 1995). A higher external Ca²⁺ concentration (5 mM Ca²⁺) suppressed the K⁺ transport defect exhibited by tss1 (Fig. 4B; Table II), most likely by increasing the number of active K⁺ transporters.

The addition of NH₄⁺ drastically reduced the affinity for K⁺ in wild-type tomato (Table II), indicating the inhibition of NH₄⁺-sensitive K⁺ transport systems. A reduced affinity for K⁺ induced by NH₄⁺ has been reported in other plant species (Santa-María et al., 2000). However, NH₄⁺ not only affected the K_m but also reduced the D_max of K⁺ transport at 5 mM Ca²⁺, suggesting that NH₄⁺-sensitive components account for an important proportion of the K⁺ taken by tomato seedlings in this condition.

Increasing the Ca²⁺ concentration in the presence of NH₄⁺ has two effects. First, there is a reduction in D_max both in tss1 and wild type. Because this reduction in D_max occurs only in the presence of NH₄⁺ it is likely that the increase of Ca²⁺ specifically inhibits NH₄⁺-insensitive K⁺ transporters. Second, it recovers the K⁺ affinity of tss1 to a similar value than that observed in the wild type. Thus, tss1 shows a Ca²⁺ requirement for a normal regulation of K⁺ transport systems in K⁺-starved conditions, irrespective of NH₄⁺. The effect of this latter ion demonstrates that NH₄⁺-sensitive and -insensitive transport mechanisms are involved in K⁺ uptake in tomato root cells in conditions of K⁺ starvation.

TSS1 would play an essential role in the regulation of K⁺ transport at low Ca²⁺ concentration. Thus, it is proposed that this increase in the external Ca²⁺ concentration could lead to an increase in the internal Ca²⁺ sufficient to overcome the defective activity of TSS1 and to allow a normal regulation of the activity K⁺ transporters, both NH₄⁺-sensitive and -insensitive mechanisms.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials and Growth Conditions

Seeds were surface sterilized with 10% (v/v) commercial bleach for 30 min and washed several times with sterile water. The seeds were first germinated in sterile water until radicle emergence because we found that this method improved germination uniformity. The basal agar medium used for growth curves contained 0.5% (w/v) Suc and 0.7% (w/v) agar. The medium was based on the one described by Spalding et al. (1999) and consisted of the following: 2.5 mM NaNO₃, 2.5 mM Ca(NO₃)₂, 2 mM MgSO₄, 0.1 mM NaFeEDTA, 80 µM Ca(H₂PO₄)₂, 25 µM CaCl₂, 25 µM H₃BO₄, 2 µM ZnSO₄·, 2 µM MnSO₄, 0.5 µM CuSO₄, 0.5 µM Na₂MoO₄, 0.01 µM CoCl₂, 2.5 mM KCl (unless otherwise indicated), and 2.5 mM MES. The pH of the mixture was adjusted to 5.7 with NaOH and autoclaved for 10 min. The K⁺-and Ca²⁺-free medium consisted of the same components with the exception of K⁺ and Ca²⁺ salts. NH₄⁺ was added as NH₄NO₃. The NO₃^- concentration was compensated by adding NaNO₃. The K⁺ and Ca²⁺ were as KCl and CaCl₂·2H₂O, respectively. Different salinity conditions used in this work were obtained by adding the appropriate amount of NaCl and LiCl to the molten basal medium. Seedlings were grown under a 16-h-light: 8-h-dark photoperiod at 50 µmol m^-2 s^-1 and 70% relative humidity.

Growth Measurements

Ten seeds were used per treatment, and three replicates were run for each treatment. Increases in root length were measured with a ruler after 2 d of treatment as described by Borsani et al. (2001). The only modification was in the experiments with low K⁺, in which case the root tip was marked 2 d after transfer, and growth was measured after 3 d.

Electrophysiological Experiments

To investigate K⁺ transport at low K⁺, wild-type and tss1 seeds were surface sterilized, germinated in distilled water, and grown for 6 d in K⁺-deficient assay medium containing 10 mM MES-Bis Tris propane and 1 mM NaCl (pH 5.7). To study the effect of Ca²⁺ on K⁺ uptake at low K⁺, this medium was supplemented with 1 or 5 mM CaCl₂. To analyze the effect of NH₄⁺ on K⁺ uptake, 1 mM NH₄Cl was added to the assay media described above.

Membrane potentials (E_m) were measured using the standard glass microelectrode technique (Felle, 1981; Fernández et al., 1999). Excised roots were mounted in a Plexiglas chamber (1.1-mL volume). Continuous perfusion of the assay medium was maintained at a flux of approximately 10 mL min^-1. Epidermal root cells were impaled with single barrel microelectrodes inserted into root hairs approximately 5 to 10 mm from the apex, as described previously (Borsani et al., 2001). Increasing K⁺ concentrations ranging from 0.1 µM to 10 mM were added sequentially to characterize the K⁺ uptake in wild-type and tss1 root cells.

	ACKNOWLEDGMENTS

 
We thank Jesús Cuartero for excellent technical assistance. We also thank Alonso Rodriguez-Navarro, José Botella, and our laboratory colleagues for helpful discussions and critical reading of the manuscript.

Received July 26, 2003; returned for revision August 31, 2003; accepted September 22, 2003.

	FOOTNOTES

 
¹ This work was supported by the Ministry of Science and Technology (Spain; grant nos. BIO2002-04541-C02-01 and BOS2001-1855) and by the Junta de Andalucía (grant no. ACC.COOR.2001 RNM 176).

² These authors contributed equally to the paper.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030361.

^* Corresponding author; e-mail mabotella{at}uma.es; fax 34-95-2132000.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Borsani O, Cuartero J, Fernandez JA, Valpuesta V, Botella MA (2001) Identification of two loci in tomato reveals distinct mechanisms for salt tolerance. Plant Cell 13: 873-887[Abstract/Free Full Text]

Borsani O, Cuartero J, Valpuesta V, Botella MA (2002) Tomato tos1 mutation identifies a gene essential for osmotic tolerance and abscisic acid sensitivity. Plant J 32: 905-914[CrossRef][Web of Science][Medline]

Britto DT, Siddiqi MY, Glass AD, Kronzucker HJ (2001) Futile transmembrane NH4⁺ cycling: a cellular hypothesis to explain ammonium toxicity in plants. Proc Natl Acad Sci USA 98: 4255-4258[Abstract/Free Full Text]

Demidchik V, Davenport RJ, Tester M (2002) Nonselective cation channels in plants. Annu Rev Plant Biol 53: 67-107[CrossRef][Medline]

Epstein E (1998) How calcium enhances plant salt tolerance. Science 280: 1906-1907[Free Full Text]

Evans NH, McAinsh MR, Hetherington AM (2001) Calcium oscillations in higher plants. Curr Opin Plant Biol 4: 415-420[CrossRef][Web of Science][Medline]

Felle H (1981) A study of the current-voltage relationships of electrogenic active and passive membrane elements in Riccia fluitans. Biochim Biophys Acta 646: 151-160[Medline]

Fernández JA, García-Sánchez MJ, Felle H (1999) Physiological evidence for a proton pump and sodium exclusion mechanisms at the plasma membrane of the marine angiosperm Zostera marina L. J Exp Bot 50: 1763-1768[Abstract/Free Full Text]

Halfter U, Ishitani M, Zhu JK (2000) The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. Proc Natl Acad Sci USA 97: 3735-3740[Abstract/Free Full Text]

Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51: 463-499[CrossRef][Web of Science]

Hirsch RE, Lewis BD, Spalding EP, Sussman MR (1998) A role for the AKT1 potassium channel in plant nutrition. Science 280: 918-921[Abstract/Free Full Text]

Ishitani M, Liu J, Halfter U, Kim CS, Shi W, Zhu JK (2000) SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell 12: 1667-1678[Abstract/Free Full Text]

Kronzucker HJ, Britto DT, Davenport RJ, Tester M (2001) Ammonium toxicity and the real cost of transport. Trends Plant Sci 6: 335-337[CrossRef][Web of Science][Medline]

Liu J, Ishitani M, Halfter U, Kim CS, Zhu JK (2000) The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc Natl Acad Sci USA 97: 3730-3734[Abstract/Free Full Text]

Liu J, Zhu JK (1997) An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance. Proc Natl Acad Sci USA 94: 14960-14964[Abstract/Free Full Text]

Liu J, Zhu JK (1998) A calcium sensor homolog required for plant salt tolerance. Science 280: 1943-1945[Abstract/Free Full Text]

Maathuis FJM, Sanders D (1997) Regulation of K⁺ absorption in plant root cells by external K⁺: interplay of different plasma membrane K⁺ transporters. J Exp Bot 48: 451-458

Maathuis FJM, Amtmann A (1999) K⁺ nutrition and Na⁺ toxicity: the basis of cellular K⁺/Na⁺ ratios. Ann Bot 84: 123-133[Abstract/Free Full Text]

Malhotra B, Glass A (1995) Potassium Fluxes in Chlamydomonas reinhardtii: I. Kinetics and electrical potentials. Plant Physiol 108: 1527-1536[Abstract]

Qiu QS, Guo Y, Dietrich MA, Schumaker KS, Zhu JK (2002) Regulation of SOS1, a plasma membrane Na⁺/H⁺ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proc Natl Acad Sci USA 99: 8436-8441[Abstract/Free Full Text]

Quintero FJ, Ohta M, Shi H, Zhu JK, Pardo JM (2002) Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na⁺ homeostasis. Proc Natl Acad Sci USA 99: 9061-9066[Abstract/Free Full Text]

Reddy AS (2001) Calcium: silver bullet in signaling. Plant Sci 160: 381-404[Medline]

Rodriguez-Navarro A (2000) Potassium transport in fungi and plants. Biochim Biophys Acta 1469: 1-30[Medline]

Rubio F, Santa-María GE, Rodriguez-Navarro A (2000) Cloning of Arabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells. Physiol Plant 109: 34-43[CrossRef]

Sanders D, Brownlee C, Harper JF (1999) Communicating with calcium. Plant Cell 11: 691-706[Free Full Text]

Santa-María GE, Danna CH, Czibener C (2000) High-affinity potassium transport in barley roots: ammonium-sensitive and -insensitive pathways. Plant Physiol 123: 297-306[Abstract/Free Full Text]

Schachtman DP (2000) Molecular insights into the structure and function of plant K⁺ transport mechanisms. Biochim Biophys Acta 1465: 127-139[Medline]

Scherer HW, Mackown CT, Legget JE (1984) Potassium-ammonium uptake interactions in tobacco seedlings. J Exp Bot 35: 1060-1070[Abstract/Free Full Text]

Sentenac H, Bonneaud N, Minet M, Lacroute F, Salmon JM, Gaymard F, Grignon C (1992) Cloning and expression in yeast of a plant potassium ion transport system. Science 256: 663-665[Abstract/Free Full Text]

Shi H, Ishitani M, Kim C, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na⁺/H⁺ antiporter. Proc Natl Acad Sci USA 97: 6896-6901[Abstract/Free Full Text]

Spalding EP, Hirsch RE, Lewis DR, Qi Z, Sussman MR, Lewis BD (1999) Potassium uptake supporting plant growth in the absence of AKT1 channel activity: inhibition by ammonium and stimulation by sodium. J Gen Physiol 113: 909-918[Abstract/Free Full Text]

Vale FR, Jackson WA, Volk RJ (1987) Potassium influx into maize root systems: influence of root potassium concentration and ambient ammonium. Plant Physiol 84: 1416-1420[Abstract/Free Full Text]

Vale FR, Volk RJ, Jackson WA (1988) Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions. Planta 173: 424-431[CrossRef][Web of Science]

Véry A-A, Sentenac H (2003) Molecular mechanisms and regulation of K⁺ transport in higher plants. Annu Rev Plant Biol 54: 575-603[CrossRef][Medline]

Wang MY, Siddiqi MY, Glass AD (1996) Interactions between K⁺ and NH₄⁺: effects on ion uptake by rice roots. Plant Cell Environ 19: 1037-1046[CrossRef]

White PJ (1997) Cation channels in the plasma membrane of rye roots. J Exp Bot 48: 499-514

Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Physiol Plant Mol Biol 53: 247-273[CrossRef][Medline]

This article has been cited by other articles:

				A. L. Schapire, B. Voigt, J. Jasik, A. Rosado, R. Lopez-Cobollo, D. Menzel, J. Salinas, S. Mancuso, V. Valpuesta, F. Baluska, et al. Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability PLANT CELL, December 1, 2008; 20(12): 3374 - 3388. [Abstract] [Full Text] [PDF]

				S.-M. Wang, J.-L. Zhang, and T. J. Flowers Low-Affinity Na+ Uptake in the Halophyte Suaeda maritima Plant Physiology, October 1, 2007; 145(2): 559 - 571. [Abstract] [Full Text] [PDF]

				A. Rosado, A. L. Schapire, R. A. Bressan, A. L. Harfouche, P. M. Hasegawa, V. Valpuesta, and M. A. Botella The Arabidopsis Tetratricopeptide Repeat-Containing Protein TTL1 Is Required for Osmotic Stress Responses and Abscisic Acid Sensitivity Plant Physiology, November 1, 2006; 142(3): 1113 - 1126. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 134/1/452    most recent pp.103.030361v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Rubio, L. Articles by Botella, M. A. Search for Related Content PubMed PubMed Citation Articles by Rubio, L. Articles by Botella, M. A. Agricola Articles by Rubio, L. Articles by Botella, M. A.