INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

K⁺ plays unique and important roles in all living cells. The high K⁺ concentrations required by plants to sustain growth convert the uptake of this element by roots in a critical process in nutrient-poor environments, where K⁺ availability may be a limiting factor for plant productivity (Clarkson, 1985). K⁺ nutrition, particularly at low external K⁺ concentrations, is frequently impaired by an excess of Na⁺ or NH₄⁺ in the solution bathing the roots (Rufty et al., 1982; Flowers and Läuchli, 1983). Reciprocally, the maintenance of an adequate K⁺ concentration inside the cells is thought to play a protective role against the detrimental effects of high external Na⁺ and NH₄⁺ concentrations (Cao et al., 1993; Zhu et al., 1998). A protective role for K⁺ during the development of water deficit has been also proposed (Gupta et al., 1989). Thus, understanding the processes involved in the movement of K⁺ toward, between, and within plant cells is a central issue in studies of the resistance of higher plants to a wide panoply of environmental stresses.

Early kinetic studies performed with barley roots demonstrated that the uptake of K⁺ from low external K⁺ concentrations can be described at a phenomenological level in terms of the Michaelis-Menten equation (Epstein et al., 1963). Later kinetic studies with other plant species (Epstein, 1973) and alternative approaches (Kochian and Lucas, 1982; Maathuis and Sanders, 1994) confirmed the universal presence of this transport mechanism usually referred to as "mechanism 1" or the "high-affinity transport system." Although the precise nature of the molecular systems underlying "mechanism 1" remained elusive for more than 30 years, recent studies have suggested that members of three families of alkali cation transporters are likely to be involved in the transport of K⁺ into the root symplasm from micromolar K⁺ concentrations: AKT1 (Sentenac et al., 1992), HKT1 (Schachtman and Schroeder, 1994; Rubio et al., 1995), and the HAK-Kup transporters HvHAK1 and AtKup1 (Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998).

Recently, an insertional mutant line for AKT1 has been identified in Arabidopsis, which exhibits a conditional capacity to grow at micromolar K⁺ concentrations (Hirsch et al., 1998). This finding indicates that, at least in some environments, the AKT1 inward-rectifier K⁺ channel could be involved in the transport of K⁺ from low K⁺ concentrations in Arabidopsis. Interestingly, akt1 plants are unable to grow at low external K⁺ concentrations only if millimolar NH₄⁺ concentrations are present in the growth medium; this is an indication that other parallel NH₄⁺-sensitive pathways of K⁺ transport exist. Evidence for an inhibitory effect of NH₄⁺ on this transport process has been earlier offered for other plant species on the basis of short-term radiometric studies suggesting that NH₄⁺-sensitive and -insensitive components could be present (Deane-Drummond and Glass, 1983; Scherer et al., 1984; Vale et al., 1987, 1988; Wang et al., 1996). However, the relevance of these components to long-term K⁺ nutrition and the molecular systems contributing to each of them remain essentially unknown.

While there is some controversy regarding the role of the HKT1 K⁺/Na⁺ cotransporter in the movement of K⁺ from the external solution into root cells (Maathuis et al., 1996; Rubio et al., 1996; Walker et al., 1996b; Wang et al., 1998), the HvHAK1 transporter exhibits some of the hallmarks expected for a major contributor to high-affinity K⁺ transport in roots (Santa-María et al., 1997). Preliminary evidence indicated that the K⁺-transport activity of HvHAK1 is strongly affected by the presence of NH₄⁺. A detailed exploration of the effect of NH₄⁺ on the transport properties of HvHAK1 and HKT1 may offer further evidence on the involvement of these transporters in the NH₄⁺-sensitive and -insensitive pathways of K⁺ and Rb⁺ transport.

To date, a functional characterization in planta of the structural and regulatory elements involved in K⁺ uptake has been partially done only in Arabidopsis (Wu and Zhu, 1996; Hirsch et al., 1998; Zhu et al., 1998; Spalding et al., 1999). No comparative studies have been made to support the idea that Arabidopsis could be a universal model system to understand the physiology of K⁺ uptake in other photosynthetic organisms, particularly in monocotyledonous plants. Consequently, intensive studies are necessary to elucidate the nature of the systems contributing to the transport of K⁺ in this major class of angiosperms. To explore the relative contribution of NH₄⁺-sensitive and -insensitive components to K⁺ transport from diluted K⁺ solutions in terms of the putative underlying molecular mechanisms already known in monocotyledonous plants, we have examined the characteristics of Rb⁺ uptake in roots of barley plants grown at combined levels of NH₄⁺ and K⁺ supply and compared them with the intrinsic properties of the HvHAK1 and HKT1 transporters as displayed in a heterologous yeast background. We found that the ionic environment encountered by barley roots during growth exerts a strong influence on the activity of the components participating in Rb⁺ transport at low Rb⁺ concentrations. We also present evidence indicating that HvHAK1 may be involved in the NH₄⁺-sensitive pathway of high-affinity Rb⁺ transport in roots.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

NH₄⁺ Inhibits Barley Growth in the Absence of K⁺

With the aim of providing an overall framework for interpreting the nature of the effects of NH₄⁺ on K⁺ transport, we first studied the long-term effect of combined levels of K⁺ and NH₄⁺ supply on growth and K⁺ accumulation. In several plant species, growth is severely inhibited by NH₄⁺ in the absence of K⁺ (Barker et al., 1967; Cao et al., 1993), while the addition of small quantities of the latter cation suppress some of the toxic effects (Cao et al., 1993). Figure 1A shows that a synergistic effect of K⁺ starvation and high NH₄⁺ supply is operative in barley as well, since we found that growth in this plant species is negatively affected after 10 d of exposure to 5 mM NH₄⁺ in the absence of K⁺, while for plants grown in the presence of just 100 µM K⁺ the growth rate was similar to that measured in plants not exposed to high NH₄⁺ concentrations. In addition to this known effect on plant growth, the presence of 5 mM NH₄⁺ strongly interfered with the absorption of K⁺ by roots as well as with K⁺ transport to shoots (Fig. 1B).

View larger version (47K): [in this window] [in a new window]   Figure 1.   Long-term exposure to high external NH4+ concentrations interferes with growth, K+ accumulation, and Rb+ uptake in barley. The effect of combined levels of K+ and NH4+ on the whole plant relative growth rate (RGR) (A), the specific absorption and translocation rates of K+ (SARK and STRK, respectively) (B), Rb+ uptake into roots 3 and 10 d after the beginning of the treatments (C), and the concentration of K+ in roots (D). RGR, SARK, and STRK were determined from the data corresponding to the period between 0 and 10 d after the beginning of the treatments. The external K+ concentration used for growth was 0 or 100 µM (white and black symbols in D, respectively), while the external NH4+ concentration was 0 or 5 mM (squares and inverted triangles in D, respectively). Measurements of Rb+ uptake were made at 100 µM Rb+ in a solution of the same composition as used for growth, except for the absence of K+. Results are average values of an experiment consisting of four independent replicates. The presence of the same letter indicates the absence of significant differences among treatments at P = 0.05.

Next, we investigated the effect of combined levels of K⁺ and NH₄⁺ on the uptake of Rb⁺(⁸⁶Rb) in barley roots measured in the same conditions as used for growth, except for the exclusion of K⁺ from the loading solution. Figure 1C shows that after 3 or 10 d of exposure to combined levels of NH₄⁺ and K⁺, the uptake of Rb⁺(⁸⁶Rb) in roots of K⁺-starved plants not exposed to high external NH₄⁺ concentrations was higher than that measured for plants grown in the presence of 100 µM K⁺ in either the absence or presence of NH₄⁺. Remarkably, the high uptake of Rb⁺ observed in K⁺-starved plants was not observed when 5 mM NH₄⁺ was included in the culture solution. This effect was not linked with the bulk concentration of K⁺ in roots, which declined markedly for K⁺-starved plants grown in the presence or the absence of NH₄⁺ (Fig. 1D).

To explore to what extent the pattern described above depends on the time of exposure and the external supply of NH₄⁺, the time course of Rb⁺(⁸⁶Rb) uptake by 14-d-old plants suddenly exposed to 0, 0.15, or 5 mM NH₄⁺ in the presence or the absence of K⁺ was also studied. Figure 2 shows that while a low NH₄⁺ concentration did not exert a strong effect, a high NH₄⁺ concentration immediately inhibited the transport of Rb⁺. The inhibition due to 5 mM NH₄⁺ was also evident 6 h after exposure to the new external K⁺ and NH₄⁺ concentrations. However, the long-term effect of NH₄⁺ on Rb⁺ transport depended on whether K⁺ was included in the growth medium. For K⁺-starved plants, high external concentrations of NH₄⁺ resulted in a low uptake of Rb⁺ within the first 3 d of treatment. However, for plants grown in the presence of 100 µM K⁺, the inhibitory effect of external NH₄⁺ was almost nil at the end of this period. These results support the notion that plants grown in the presence of high external NH₄⁺ concentrations are able to overcome the initial inhibitory effect. Note that for plants always kept in a constant ionic environment, the uptake of Rb⁺ declined along the developmental window studied. A decline in K⁺ uptake during plant ontogeny has been previously observed in other Triticeae species (Kuhlmann and Barraclough, 1987) and is likely to be the result of a regulatory process superimposed on that studied here.

View larger version (19K): [in this window] [in a new window]   Figure 2.   NH4+ effect on Rb+ uptake depends on the time of exposure as well as on the presence of K+ in the growth medium. Time course of Rb+(86Rb) uptake in plants exposed to the presence (black symbols) or the absence (white symbols) of 100 µM K+ at different NH4+ concentrations: squares, 0 mM; triangles, 0.15 mM; and inverted triangles, 5 mM NH4+. Measurements were made at 100 µM Rb+ 0, 6, and 66 h after the beginning of the treatments. The composition of the loading solution was the same as used for each growth condition, except for the exclusion of K+. Results are from an experiment consisting of three independent replicates. The presence of the same letter for a given time of exposure to different treatments indicates the absence of significant differences at P = 0.05.

Long-Term Exposure to High NH₄⁺ Levels Elicits a Component of Rb⁺ Uptake Insensitive to NH₄⁺

The results above suggest the possibility that different systems may participate in the transport of Rb⁺ into roots after a long-term exposure to different external concentrations of K⁺ and NH₄⁺. To test this hypothesis, we studied the effect of Na⁺ and NH₄⁺ on the uptake of Rb⁺ in 17-d-old plants exposed for the last 3 d to combined levels of K⁺ and NH₄⁺. Preliminary experiments showed that the inclusion of Na⁺ in the growth medium did not affect markedly the pattern of Rb⁺ uptake described above (data not shown). The inclusion of 100 µM Na⁺ in the growth treatments allowed the possibility that some transport systems might require the presence of this cation to be operative.

Figure 3A shows that in plants exposed for 3 d to the presence of NH₄⁺ and the absence of K⁺, the uptake of Rb⁺ measured in the absence of NH₄⁺ did not reach the high values observed in plants grown in the absence of both K⁺ and NH₄⁺ (Fig. 3B), which indicates that long-term NH₄⁺ nutrition interferes with a pathway responsible for the high rate of Rb⁺ transport observed after K⁺ starvation. Aside from this absolute difference, the relative effect of NH₄⁺ on Rb⁺ uptake was also dependent on the external concentrations of K⁺ and NH₄⁺ to which the roots had been exposed. Figure 3, B and D, shows that for plants grown in the absence of NH₄⁺, Rb⁺ uptake was sharply inhibited by the inclusion of 5 mM NH₄⁺ in the loading solution, while Na⁺ did not exert any significant effect. The inhibitory effect of NH₄⁺ was more pronounced in plants grown in the absence than in the presence of K⁺ (Fig. 3B). A separate experiment showed that Rb⁺ uptake by plants grown in the absence of NH₄⁺ was reduced to 58% and 60% by addition of 100 µM K⁺ to K⁺-starved and K⁺-sufficient plants, respectively (data not shown).

View larger version (57K): [in this window] [in a new window]   Figure 3.   NH4+ exerts a differential effect on the transport of Rb+ in barley plants grown in different ionic environments. The short-term effect of 5 mM NH4+, in combination with the presence or the absence of 100 µM Na+, on the uptake of Rb+ was determined at 100 µM Rb+ for plants previously exposed for 3 d to the absence of K+ in the presence of 5 mM NH4+ (A), the absence of both K+ and NH4+ (B), 100 µM K+ in the presence of 5 mM of NH4+ (C), and 100 µM K+ in the absence of NH4+ (D). All growth solutions contained 100 µM Na+. Note the use of a different scale in B. Results given are the average values of two separate experiments consisting of three and five independent replicates. For each growth condition, the presence of the same letter on two columns indicates the absence of significant differences at P = 0.05.

Conversely, in plants grown in the presence of 5 mM NH₄⁺, the presence of 5 mM NH₄⁺ in the loading solution led to a relatively small decrease of Rb⁺ uptake, and Na⁺ did not produce a significant effect (Fig. 3, A and C). Thus, for plants grown in the absence of NH₄⁺, Rb⁺ uptake is dominated by a component sensitive to NH₄⁺, whereas in plants grown in the presence of NH₄⁺, the bulk uptake of Rb⁺ is mediated by a component able to operate in the presence of high NH₄⁺ concentrations. Complementary experiments showed that Rb⁺ uptake for plants grown at 5 mM NH₄⁺ in the presence or absence of K⁺ was reduced to 78% and 94%, respectively, by the addition of 100 µM K⁺ (data not shown). To obtain some additional information on the systems contributing to Rb⁺ uptake in plants grown in the presence of NH₄⁺, we also studied the effect of CsCl and BaCl₂ on Rb⁺ uptake. While the addition of 100 µM CsCl produced a 27% inhibition, the addition of 1 mM BaCl₂ resulted in a 39% inhibition of Rb⁺ transport into roots (Fig. 4).

View larger version (30K): [in this window] [in a new window]   Figure 4.   Cs+ and Ba2+ inhibit Rb+ uptake in plants grown in the absence of K+ and the presence of 5 mM NH4+. Relative effect of CsCl (100 µM) and BaCl2 (1 mM) on the uptake of Rb+(86Rb) in plants exposed for 3 d to the absence of K+ and the presence of both 5 mM NH4+ and 100 µM Na+. Rb+(86Rb) uptake was measured at 100 µM Rb+ in the absence of NH4+ and the presence of 100 µM Na+. On the left, the uptake of Rb+(86Rb) measured in the absence of inhibitors is shown. Results are from an experiment consisting of five independent replicates. The presence of the same letter on two columns indicates the absence of significant differences at P = 0.05.

Yeast Cells Expressing HKT1 Exhibit an Enhanced Resistance to High External NH₄⁺ Concentrations Compared with Those Expressing HvHAK1

Yeast cells of the strain W3, which are defective in high-affinity K⁺ transport, were used to explore the effect of external NH₄⁺ on the transport of Rb⁺ mediated by the HKT1 and HvHAK1 transporters. Figure 5A shows that W3 cells did not grow at low external K⁺ concentrations regardless of the external NH₄⁺ concentration supplied to the medium. Transformation of these cells with HKT1 or HvHAK1 restored growth both at 10 (Fig. 5A) and 100 (data not shown) µM K⁺ in the absence of NH₄⁺. A progressive inhibition of growth was observed for HvHAK1-expressing cells at increasing levels of NH₄⁺, more so at 10 than at 100 µM K⁺. At 10 µM K⁺ in the presence of 50 or 100 mM NH₄⁺, these cells were unable to grow or grew poorly. However, under the same conditions, the growth of yeast cells expressing HKT1 was only slightly reduced. These results indicate that the expression of HKT1 conferred on W3 cells an enhanced capacity to grow at very high external NH₄⁺ concentrations relative to that exhibited by yeast cells expressing HvHAK1; furthermore, the inhibition of growth in yeast cells expressing HvHAK1 depended markedly on the balance between the external concentrations of K⁺ and NH₄⁺. To explain these findings in terms of the kinetic properties of HKT1 and HvHAK1, we examined the dependence of Rb⁺(⁸⁶Rb) uptake on the external NH₄⁺ concentration displayed by these transporters. Figure 5B shows that the uptake of Rb⁺(⁸⁶Rb) mediated by HvHAK1 decreased with the increase in external NH₄⁺ concentrations, with a half inhibition at 2.7 mM NH₄⁺. At 5 mM NH₄⁺, Rb⁺(⁸⁶Rb) uptake was inhibited to 27% of that measured in the absence of NH₄⁺. Interestingly, the uptake of Rb⁺(⁸⁶Rb) in yeast cells expressing HKT1 was sharply inhibited by external NH₄⁺ concentrations considerably lower than those required to inhibit Rb⁺(⁸⁶Rb) uptake mediated by HvHAK1 to the same extent; it was almost nil at 5 mM NH₄⁺ (Fig. 5C).

View larger version (34K): [in this window] [in a new window]   Figure 5.   HKT1 confers on yeast cells an enhanced capacity for growth in the presence of high external NH4+ concentrations. A, Growth of W3 yeast cells, defective in K+ uptake, and the same cells transformed with HvHAK1 or HKT1 on AP medium supplied with K+ 10 µM at two levels of NH4+ addition; 10 µL of progressive dilutions of a 0.1-optical density cell suspension were inoculated on the medium. B, Rb+(86Rb) uptake mediated by W3 cells expressing HvHAK1 at 100 µM Rb+ at increasing levels of NH4+. C, As in B, but for HKT1-expressing W3 cells. In B and C, results are the average values of not less than three experiments. D, Results of a representative experiment showing the accumulation of K+ measured as K+ removal from a 100 µM K+ solution mediated by HKT1-expressing cells in the presence (white symbols) or absence (black symbols) of 5 mM NH4+.

When the results obtained in the growth and Rb⁺(⁸⁶Rb)-uptake experiments are compared, a consistent pattern emerges for HvHAK1, but a contradiction is apparent for HKT1. This anomaly can be explained if the uptake of Rb⁺ does not mirror the uptake of K⁺ mediated by HKT1 in the presence of high external NH₄⁺ concentrations. Figure 5D shows that the accumulation of K⁺ by HKT1-expressing cells was only slightly lower in the presence of 5 mM NH₄⁺ than in the absence of this cation. This pattern contrasts with that observed in HvHAK1-expressing cells, for which the K⁺ accumulation was sharply reduced by 5 mM NH₄⁺ (Santa-María et al., 1997). Thus, HKT1 is able to mediate the uptake of K⁺ even at high external NH₄⁺ concentrations, whereas K⁺ uptake mediated by HvHAK1 is only marginal under these conditions. The slow growth rate and slow net K⁺ uptake of HKT1-expressing yeast cells can be explained by the strong depolarization induced by HKT1 in yeast (Madrid et al., 1998).

NH₄⁺ Exerts a Mixed Inhibitory Effect on HvHAK1-Mediated Rb⁺ Transport

Because of the possibility that HvHAK1 is involved in an NH₄⁺-sensitive component of Rb⁺ transport in roots, we further examined the effect of the former cation on the kinetic parameters of Rb⁺ transport mediated by HvHAK1, as determined in W3 yeast cells (Fig. 6A). Estimations of V_max and apparent K_m from the data shown in Figure 6A yielded the following values: 6.54, 4.97, and 3.11 nmol mg¹ min¹ and 29.4, 140, and 213 µM Rb⁺ at 0, 2.5, and 5.0 mM NH₄⁺, respectively. This repeated effect on both V_max and K_m values suggests the presence of a mixed inhibition kinetics. Repeated attempts to complement the growth of mep1 mep2 mep3 yeast cells, which are defective in NH₄⁺ transport, with the HvHAK1 cDNA failed. We were also unable to observe any increase in methylamine uptake between mep1 mep2 mep3 cells and the cells expressing HvHAK1 at 1 mM methylamine (data not shown). These results indicate that HvHAK1 could not transport NH₄⁺ and that Rb⁺ uptake inhibition by NH₄⁺ could not imply competition between these ions for the entry into the pore domain. Equally importantly, we observed that this inhibition was reversed by NH₄⁺ removal (Fig. 6B).

View larger version (26K): [in this window] [in a new window]   Figure 6.   Rb+ transport mediated by HvHAK1 is reversibly affected by NH4+ through a mixed mode inhibition. A, Uptake of Rb+(86Rb) by W3 cells expressing HvHAK1 in the absence () or presence of 2.5 () or 5 () mM NH4+. B, Immediate effect of NH4+, and the effect of NH4+ removal after a 15-or 30-min exposure to 5 mM NH4+ (right side) on Rb+ uptake measured from a 100 µM Rb+ solution; on the left, the rate of Rb+ measured in cells never exposed to NH4+ is shown. Results shown in A and B are the average values of five and three experiments, respectively. The presence of the same letter on two columns indicates the absence of significant differences at P = 0.05.

A previous report (Santa-María et al., 1997) showed that the uptake of Rb⁺ by W3 cells expressing HvHAK1 is inhibited by K⁺ (K_i = 27 µM) and Na⁺ (K_i = 15 mM). In addition to exploring the inhibitory effect of NH₄⁺, we studied the effect of other monovalent cations on Rb⁺ uptake mediated by HvHAK1. We found that CsCl inhibited the uptake of Rb⁺ (K_i = 80 µM), while the addition of LiCl did not affect this transport process (data not shown). Thus, the inhibitory effect of monovalent cations on Rb⁺ uptake mediated by HvHAK1 follows the sequence K⁺>Cs⁺> NH₄⁺Na⁺, with Li⁺ exerting no effect, at least within the range of external concentrations studied here. We also examined the effect of Ba²⁺ on the transport of Rb⁺ mediated by HvHAK1 at 100 µM Rb⁺, and found that concentrations of BaCl₂ much higher than those inhibiting Rb⁺ uptake in plants grown at 5 mM NH₄⁺ were necessary to produce a detectable inhibition of Rb⁺ uptake in HvHAK1-expressing yeast cells, with a half inhibition at 16 mM (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The identity and contribution of the systems involved in the transport of K⁺ from dilute K⁺ solutions have been extensively discussed in the recent literature (Schachtman and Liu, 1999; Rodríguez-Navarro, 2000). Because of the difficulties derived from the use of ⁴²K⁺, most of the studies referring to the unidirectional fluxes of K⁺ in roots have been done with Rb⁺(⁸⁶Rb) instead of K⁺(⁴²K) or with ⁸⁶Rb as a tracer for K⁺. It is worth noting that Rb⁺ can be used as a K⁺ analog only under certain circumstances (Rodríguez-Navarro, 2000). For some transport systems, even such simple characteristics as the NH₄⁺ sensitivity of the uptake can be different for Rb⁺ and K⁺, as clearly illustrated in Figure 5. In this report, we have used Rb⁺ to characterize some of the root-expressed transporters of monocotyledonous plants, depending on whether they mediate NH₄⁺-sensitive or -insensitive Rb⁺ uptake. Because of the existence of differences in the rates of K⁺ and Rb⁺ transport, extrapolation of Rb⁺ measurements to the transport of K⁺ in roots should be done with caution.

The notion that plant roots are furnished with NH₄⁺-sensitive and -insensitive components for the transport of K⁺ and Rb⁺ from diluted solutions into the root symplasm of higher plants was developed previously (Deane-Drummond and Glass, 1983; Scherer et al., 1984; Vale et al., 1987, 1988). However, the contribution of these components to plant K⁺ nutrition has not been deeply explored. Here we present evidence that the contribution made by these components to the uptake of Rb⁺ depends on the external concentration of K⁺ and NH₄⁺ to which the roots were exposed during growth (Figs. 1-3). We observed that for barley plants grown in the absence of NH₄⁺ with an adequate provision of nitrogen, Rb⁺ uptake was extremely sensitive to NH₄⁺. This result is consistent with those previously reported by Wang et al. (1996) for rice plants grown in the absence of or at low external NH₄⁺ concentrations in a medium containing 1.5 mM NO₃. In addition, we found that in barley plants grown at high NH₄⁺ concentrations, Rb⁺ uptake is preferentially mediated by a system (or systems) insensitive to NH₄⁺.

It has recently been argued that in Arabidopsis, the NH₄⁺-sensitive component of K⁺ transport can be attributed to HKT1 and/or Kup-HAK transporters (Spalding et al., 1999). However, no critical information has been available to test this hypothesis properly, while the NH₄⁺-insensitive component is thought to correspond to the AKT1 K⁺ channel (Hirsch et al., 1998). Whether these systems are also involved in the transport of Rb⁺ from micromolar Rb⁺ concentrations in monocotyledonous plants has not been examined. A comparison of the properties exhibited by the main types of root-expressed Rb⁺ transporters in heterologous systems with those observed in roots helped us to make an assessment of the identity of the systems involved in each component of Rb⁺ transport in barley, and also to test the relevance of HKT1 and HAK1 transporters in the NH₄⁺-sensitive component.

NH₄⁺-Sensitive Systems of Rb⁺ Transport

Several lines of evidence obtained in this work converge in assigning to HvHAK1 a major role in high-affinity Rb⁺ transport in roots of plants grown in the absence of K⁺ and NH₄⁺. The absence of an effect of Na⁺ and the inhibitory effect of external K⁺ and NH₄⁺ on Rb⁺ transport in roots of plants grown under these conditions (Epstein et al., 1963; Vale et al., 1987, 1988; Fig. 3B) and HvHAK1-expressing yeast cells (Santa-María et al., 1997; Fig. 5B) offer evidence supporting this possibility. In the same way, the mixed inhibitory effect of NH₄⁺ on Rb⁺ transport kinetics, as well as recovery of transport after NH₄⁺ removal, has been observed in maize and tobacco roots (Breteler, 1977; Scherer et al., 1984) and in HvHAK1-expressing yeast cells (Fig. 6). Furthermore, the inhibitory effect of monovalent cations on the transport of Rb⁺ mediated by HvHAK1 reported here follows a sequence similar to that previously determined in barley roots and maize shoots of plants grown in the absence of NH4⁺ (Epstein et al., 1963; Smith and Epstein, 1964). The intrinsic properties of HvHAK1 observed in W3 yeast cells seem to be consistent with a major role of this transporter in the NH₄⁺-sensitive component of high-affinity Rb⁺ transport in plants grown under limiting external K⁺ concentrations in the absence of or at low external NH₄⁺ concentrations. Interestingly, our results indicate that HvHAK1 does not act as an NH₄⁺ transporter.

The second possible candidate thought to be involved in the NH₄⁺-sensitive component of Rb⁺ transport is HKT1. Results shown in Figure 5C support this possibility. However, the absence of a stimulatory effect of Na⁺ on the transport of Rb⁺ (when measured in the absence of NH₄⁺) observed in plants grown at different pH in the presence or absence of Na⁺ (Maathuis et al., 1996), as well as for plants grown at different external concentrations of K⁺ and NH₄⁺ (Fig. 3), indicate that HKT1 does not contribute, or contributes only as a minor system, to the total uptake of Rb⁺ into barley roots regardless of the ionic conditions prevailing in the external solution in which plants had been grown. Remarkably, while HKT1 acts as an NH₄⁺-sensitive pathway of high-affinity Rb⁺ transport, it operates as an NH₄⁺-insensitive pathway of K⁺ accumulation inside the yeast cells (Fig. 5). In akt1 Arabidopsis plants, changes in membrane potential associated with the transport of K⁺ were greatly enhanced by Na⁺ and reduced by NH₄⁺ addition (Spalding et al., 1999). This result and those reported here indicate that no inhibition of K⁺ accumulation should be observed after NH₄⁺ addition if the HKT1 identified in the Arabidopsis genome behaves as its wheat counterpart in yeast cells. It should be mentioned that because of the existence of differences in the rates of K⁺ and Rb⁺ transport mediated by HKT1 (Gassmann et al., 1996), the possibility that HKT1 is a contributor to the NH₄⁺-insensitive component of K⁺ accumulation in barley roots could be not rejected.

The NH₄⁺-Insensitive Pathway of Rb⁺ Transport

The heterologous studies reported above clearly indicate that neither HKT1 nor HvHAK1 were involved in the component of Rb⁺ transport insensitive to NH₄⁺. Thus, the next question refers to the identity of the systems involved in that pathway. Studies made with yeast and insect cells expressing the inward-rectifier K⁺ channel AKT1 have shown that it is about 10- to 20-fold more permeable to K⁺ than to NH₄⁺ (Bertl et al., 1997; C. Horeau, personal communication). These observations and those derived from the null mutant of AKT1 in Arabidopsis (Hirsch et al., 1998) suggest the possibility that a barley AKT1 could be responsible for the NH₄⁺-insensitive component observed here. The presence of AKT1 in monocot genomes has been previously established (Hoth et al., 1997; accession no. Y07632.1), and electrophysiological studies suggest the involvement of an AKT1-like channel in low-affinity K⁺ transport into barley roots (Amtmann et al., 1999). However, compelling evidence indicates that passive movement of K⁺ from the external solution into the cytosol of epidermal and cortical root cells of this plant species only takes place when the external K⁺ concentration is higher than 0.5 mM (Walker et al., 1996a), which rules out the involvement of K⁺ channels in the transport of K⁺ from dilute K⁺ solutions (Kochian and Lucas, 1993; Maathuis and Sanders, 1993, 1994).

To date, no information is available to determine if a similar thermodynamic impediment also applies to plants grown in the presence of high external NH₄⁺ concentrations. Thus, the only way to make an assessment of the possible contribution of AKT1 to the NH₄⁺-insensitive component is to compare the properties exhibited by heterologous systems expressing AKT1 with those observed in barley plants. The absence of an effect of Na⁺ on Rb⁺ transport in roots is consistent with the selectivity properties displayed by both HvHAK1 and AKT1, while the weak effect of K⁺ on Rb⁺ transport could be explained either by a high Rb⁺ K_m and/or a high K⁺ K_i. Therefore, these two properties serve to characterize the NH₄⁺-insensitive component, but are not useful to explore the possible involvement of AKT1 in this pathway. However, studies with yeast cells expressing AKT1 have shown that inward K⁺ currents mediated by this channel are strongly blocked by Ba²⁺ and are abolished by very low Cs⁺ concentrations (Bertl et al., 1997). In the present study, we found that while the effect of 1 mM BaCl₂ could be consistent with a role of AKT1 in this process, the effect of 100 µM CsCl on Rb⁺ transport was much lower than expected if this hypothesis were correct (Fig. 4). In fact, the slight reduction of Rb⁺ transport observed in barley roots upon the addition of CsCl could be explained by the inhibitory effect exerted by Cs⁺ on other transporters (such as HvHAK1) potentially involved in the small NH₄⁺-sensitive component of Rb⁺ transport of NH₄⁺-grown plants. Thus, none of the transport systems expressed in roots to date characterized exhibit intrinsic properties entirely consistent with those displayed by the NH₄⁺-insensitive component of Rb⁺ transport in barley, and, consequently, the identity of the systems contributing to this pathway could be not resolved. Therefore, the possibility that other non-characterized root-expressed K⁺ transporters (Rubio et al., 2000) participate in this kinetic pathway could not be excluded.

The idea that several overlapping or partially redundant systems participate in the transport of K⁺ or Rb⁺ into the root symplasm from micromolar K⁺ or Rb⁺ concentrations was initially discussed by Walker et al. (1996b) and Rubio et al. (1996) and received further support for dicotyledonous plants from the disruption of AKT1 in Arabidopsis (Hirsch et al., 1998). Our results provide evidence that this concept may be applied for the transport of Rb⁺ to monocotyledonous plants, although the identity of the major systems contributing to the NH₄⁺-sensitive and -insensitive pathways could be different in Arabidopsis and barley.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Growth

Seeds of barley (Hordeum vulgare cv Golden promise) were sown on moistened filter paper and kept in the dark for 48 h. Seedlings were then transferred to an acrylic ring, and placed on a 0.8-L plastic pot containing a nutrient solution of the following composition: 1.0 mM Ca(NO₃)₂, 0.5 mM MgSO₄, 0.5 mM H₃PO₄, 50 µM FeEDTA, 50 µM CaCl₂, 25 µM H₃BO₃, 2 µM ZnSO₄, 2 µM MnSO₄, 0.5 µM CuSO₄, 0.5 µM molybdic acid, 2.5 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and 100 µM KCl. The pH was brought to 6.00 ± 0.05 by the addition of Ca(OH)₂. To avoid the possibility that the low external K⁺ concentrations supplied could be limiting for plant growth, the growth rate was reduced by manipulating the photon flux density at the plant level. It was set at 70 µmol m² s¹ over a photoperiod of 16 h. The temperature in the growth chamber was set to 22°C (day) and 18°C (night). Relative humidity was kept at 85%, day and night. The nutrient solution, permanently aerated, was renewed every 3 d during the first week and then every 2 d until the beginning of the experiments on d 14 after germination. During the afternoon of d 13, the solution was again renewed and the experiments started 3 h after the beginning of the light period of d 14. During the course of the experiments the solution was renewed daily. For measurements of growth and chemical analysis, four plants were harvested before and after each treatment, divided into shoots and roots, and weighed. Subsequently, samples were placed in plastic vials and treated with HCl 0.5 N to allow the release of free cations. The concentration of K⁺ was determined in dilutions of the extracts with atomic absorption spectrophotometer on emission mode (Shimadzu, Columbia, MD). The plant relative growth rate and the specific absorption and translocation rates of K⁺ were estimated according to methods previously described (Santa-María and Cogliatti, 1998).

Measurements of Rb⁺(⁸⁶Rb) Uptake in Plants

Because some plant K⁺ transporters strongly discriminate K⁺ over Rb⁺, we used ⁸⁶Rb as a tracer for the uptake of Rb⁺, excluding K⁺ from the loading solution. Thus, for measurements of Rb⁺(⁸⁶Rb) uptake, roots of intact plants were transferred for 5 min to a pot containing the complete nutrient solution described above without K⁺. This step was performed to allow the elution of the K⁺ contained in the root apoplast. Following this treatment, plants were transferred to plastic pots containing 0.14 L of the same solution without K⁺, vigorously aerated, to which Rb⁺(⁸⁶Rb) had been previously added to reach a 100 µM Rb⁺ concentration. The loading period was set at 20 min, and was followed by two wash outs with a solution of the same ionic composition as used for loading without ⁸⁶Rb for a total of 6 min. An exception to this procedure was made when the effect of CsCl or BaCl₂ was studied; in these experiments, the wash-out solutions did not include the inhibitors. Plants were harvested as previously described and the radioactivity in the samples was determined in a liquid scintillation counter (model 1414, Wallac, Turku, Finland).

Yeast Growth Complementation Tests and Uptake Experiments

Studies of complementation of W3 yeast cells (trk1 trk2) were done in a mineral solid medium with Arg as the basic nitrogen source, and supplemented with different amounts of K⁺ and NH₄⁺ provided as chloride salts. W3 cells were transformed either with a pYPGE15 plasmid containing the HvHAK1 cDNA under the control of the PGK1 promoter (Santa-María et al., 1997) or with a pDR195 plasmid containing the wheat HKT1 cDNA under the control of the PMA1 promoter (Rubio et al., 1995). For complementation studies, 10 µL of a series of dilutions of 0.1 absorbance cell suspensions of W3 cells and W3 cells expressing HvHAK1 or HKT1 were placed in each plate. For Rb⁺(⁸⁶Rb) uptake experiments, cells were grown overnight in Arg medium plus 40 mM KCl, and were K⁺ starved for 4 h before the start of the experiments. These experiments were conducted on MES/Ca²⁺, pH 6.0, for HvHAK1-expressing or MES/Ca²⁺ plus 0.5 mM NaCl for HKT1-expressing yeast cells. Other conditions, as well as the procedure followed in experiments done to study K⁺ removal by HKT1 expressing yeast cells from mineral medium supplemented with 100 µM K⁺, were the same as described elsewhere (Santa-María et al., 1997).

Complementation studies with the yeast strain 31019 (mep1 mep2::LEU2 mep3::KanMX2 ura3; Marini et al., 1997), which is defective in NH₄⁺ transport, were in a mineral medium, without Arg and containing Na₂HPO₄/NaH₂PO₄ (pH 6.0). The medium was supplemented with 20 µM KCl at increasing NH₄Cl concentrations as the sole source of nitrogen. Uptake of methylamine (¹⁴CH₃NH₂) in these cells was measured following a similar experimental procedure as described above for the uptake of Rb⁺.