JXB Advance Access originally published online on April 4, 2005
Journal of Experimental Botany 2005 56(416):1553-1562; doi:10.1093/jxb/eri150
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RESEARCH PAPER |
High-affinity K+ uptake in pepper plants
Departamento de Nutrición Vegetal, Centro de Edafología y Biología Aplicada del Segura-CSIC, Apartado de Correos 164, E-30100 Murcia, Spain
* To whom correspondence should be addressed. Fax: +34 968 396 213. E-mail: frubio{at}cebas.csic.es
Received 30 November 2004; Accepted 1 March 2005
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Abstract |
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High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of
and starved of K+ show an -sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 µM. When plants are grown in the presence of 
high-affinity K+ uptake is not inhibited by . Although -grown plants deplete external K+ below 1 µM in the absence of when 1 mM is present they do not deplete external K+ below 10 µM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the -sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the -sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of . The -insensitive K+ uptake may be mediated by an AKT1-like K+ channel. Key words: Ammonium, pepper, potassium, transport
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The most abundant cationic component of plant cells is K+, a macronutrient that composes up to 10% of the total plant dry weight. It fulfils important functions and its concentration is maintained constant in the cytosol around 100 mM. To acquire K+, plant roots explore the soil where they encounter solutions of many different K+ concentrations. The different compositions of the soil solutions that root cells may encounter do not preclude, however, that roots take up K+. The plasma membrane of root cells is furnished with selective K+ transporters that secure K+ supply under many different conditions, including a wide range of external K+ concentrations and the presence of other ions that interfere with K+ uptake. At low K+ concentrations in the soil solution, which are found in many agricultural soils, the function of high-affinity K+ uptake transporters is crucial to sustain plant growth and productivity (Clarkson, 1985
). In addition, an excess of Na+ or in the soil solution may impair plant growth, especially at low external K+ concentrations (Rufty et al., 1982; Flowers and Läuchli, 1983).
The first high-affinity K+ uptake system to be kinetically characterized was from barley (Hordeum vulgare) and it was shown that there was a Km for K+ of 18 µM, no discrimination between K+ and Rb+ and a low affinity for Na+ (Epstein et al., 1963). Cs+ and were shown competitively to inhibit high-affinity K+ uptake (Epstein and Hagen, 1952; Smith and Epstein, 1964). Similar systems have also been described in other plant species (Epstein, 1973; Kochian and Lucas, 1982; Maathuis and Sanders, 1994). Further studies on the regulation of K+ absorption into barley roots showed that there is a rapid up-regulation of high-affinity K+ uptake when the exogenous K+ supply is interrupted (Glass, 1975).
It has been widely accepted that the high-affinity K+ uptake is mediated by K+ transporters while the absorption of K+ in the low-affinity range of concentrations is mediated by K+ channels (Maathuis and Sanders, 1996). Molecular approaches have led to the identification in several plant species of families of genes that encode K+ transporters and K+ channels (Rodríguez-Navarro, 2000; Very and Sentenac, 2003). Members of the KT/HAK/KUP family of K+ transporters may be involved in high-affinity K+ uptake into the roots. By heterologous expression in yeast it has been shown that the barley and the rice (Oryza sativa) HAK1 transporters show characteristics which are in agreement with the high-affinity K+ uptake observed in the plant roots (Santa-María et al., 1997; Banuelos et al., 2002). They mediate Rb+ uptake with Km values for Rb+ of 18 µM for HvHAK1 and 6 µM for OsHAK1, no discrimination between Rb+ and K+, and the genes encoding these transporters are induced in the roots of K+-starved plants. Therefore, the barley and the rice HAK1 transporters are probably major contributors to high-affinity K+ uptake in these plant species. In arabidopsis (Arabidopsis thaliana), the K+ transporter HAK5 expressed in yeast also shows high-affinity for Rb+ (12.6 µM), and no discrimination between K+ and Rb+ (Rubio et al., 2000), and the gene encoding it is induced in the roots of K+-starved plants (Armengaud et al., 2004; Shin and Schachtman, 2004). On the other hand the K+ channel AKT1 of Arabidopsis is expressed constitutively (Lagarde et al., 1996) and has been described as an important component of the K+ uptake apparatus even in the high-affinity range of K+ concentrations (Hirsch et al., 1998; Spalding et al., 1999). In barley and Arabidopsis -sensitive and -insensitive components of the high-affinity K+ uptake have been described. The -sensitive component is probably mediated by a transporter of the KT/HAK/KUP family and the -insensitive by a K+ channel (Spalding et al., 1999; Santa-María et al., 2000). In relation to the -insensitive component it has been shown in barley and rice that was least inhibitory to K+ uptake if was present in the growth solution (Wang et al., 1996; Santa-María et al., 2000).
The studies performed in model plant species such as Arabidopsis or in crops such as barley or rice have provided essential and valuable information on high-affinity K+ uptake in plants, but research on other crops is needed. Pepper is an important crop and detailed physiological studies on high-affinity K+ uptake are lacking. A cDNA, CaHAK1, encoding a transporter of the KT/HAK/KUP family has recently been identified in pepper (Martínez-Cordero et al., 2004) and this transporter may be of relevance for high-affinity K+ uptake in pepper. The experiments described below examine important aspects of high-affinity K+ uptake in pepper which should be relevant for K+ nutrition of this and other crops under K+-limiting conditions and under the presence of abiotic stresses such as high concentrations or salinity. The regulation of the induction of the high-affinity K+ uptake and the effects of the growing conditions on the expression of CaHAK1 and CaAKT1, encoding a pepper homologue of the Arabidopsis AKT1, are studied. The sensitivity of the high-affinity K+ uptake to Na+, and Cs+ is characterized and the effect of the growing conditions on the sensitivity to discussed.
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Materials and methods |
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Plant growth
Seeds of pepper (Capsicum annuum L., cv. california), were prehydrated with an aerated 0.5 mM CaSO4 solution for 72 h and germinated in vermiculite at 28 °C. After 2 d the seedlings were placed in 15 l containers filled with a modified one-fifth Hoagland solution, which consisted of the following macronutrients (mM): 1.2 KNO3, 0.8 Ca(NO3)2, 0.2 KH2PO4, and 0.2 MgSO4 and the following micronutrients (µM): 50 CaCl2, 12.5 H3B03, 1 MnSO4, 1 ZnSO4, 0.5 CuSO4, 0.1 H2MoO4, 0.1 NiSO4, and 10 Fe-EDDHA. In the experiments where the effect of the presence of
in the growing solution was studied, plants were grown in a solution containing (mM): 1.4 NH4NO3, 0.2 KH2PO4, 0.2 MgSO4, 0.8 CaCl2, and 1.2 KCl, with the micronutrients described above. In the experiments where plants were grown in the presence of 100 µM K+, the solution contained (mM): 1.4 Ca(NO3)2, 0.1 Ca(H2PO4)2, 0.2 MgSO4, and 0.1 KCl and the micronutrients described above, and to avoid K+ depletion during plant growth, 120 l containers with 20 plants per container were used and the K+ in the solution monitored and corrected if needed. No significant differences were observed in the growth of plants grown with different K+ concentrations. Plants were grown in a controlled-environment chamber with a 16/8 h light/dark cycle and air temperatures of 25 °C and 20 °C, respectively. The relative humidity was 65% (day) and 80% (night) and the photon flux density 550 µmol m–2 s–1. Modest aeration was provided. The nutrient solution was replaced with fresh solution after weeks 2, 3, and 4.
K+ depletion experiments in plants and K+ concentrations in plant tissues
Four-week-old plants grown as described above were employed for K+ depletion experiments. For K+ starvation, plants were transferred for different periods of time to a nutrient solution deprived of K+ which contained (mM) 1.4 Ca(NO3)2, 0.1 Ca(H2PO4)2, and 0.2 MgSO4 and the micronutrients described above. In the experiments where the effect of the presence of in the growing solution was studied, plants were starved of K+ in a solution containing (mM) 1.4 NH4NO3, 0.2 MgSO4, 0.7 CaCl2, and 0.1 Ca(H2PO4)2, and the micronutrients described above. Control plants were maintained in the K+-containing nutrient solutions described above. Plants were rinsed in a cold K+-free nutrient solution and at time zero transferred to 250 ml containers with a K+-free nutrient solution supplemented with 50 µM KCl and different concentrations of NH4Cl, CsCl, and NaCl as indicated. Samples of 1 ml were taken at intervals for 2 h and their K+ concentrations determined by atomic emission in a Perkin-Elmer (Boston, MA) 5500 spectrophotometer. The figures show representative experiments. Data of at least five independent experiments with one plant per treatment were fitted to a Michaelis–Menten function as described elsewhere (Banuelos et al., 2002) and the Km and Vmax values calculated. Reported are average and standard error values. At the end of the experiment, plants were separated into roots and shoots and the fresh weight determined. Then, roots and shoots were dried at 65 °C for 4 d and the dry weight determined. Chemical analyses of plant organs were carried out after digestion with HNO3:HClO4 (2:1, v:v). K+ concentrations were determined by atomic absorption spectrometry. Reported are average values and error bars denote standard errors.
Split root experiments
To study the effect of subjecting one-half of the root system to K+ starvation on high-affinity K+ uptake, experiments with a split root system were performed. Plants were grown as described above for 1 month and then the root system was split into two 1.0 l containers filled with the -free solution described above, with or without K+. After 3 d, K+ depletion experiments were performed on each half of the root system, fresh and dry weights of roots and shoots determined, and chemical analyses performed as described above.
cDNA isolation and semi-quantitative RT-PCR
cDNA was synthesized from 2 µg of total RNA isolated from pepper roots by using an anchored oligo-dT primer and avian myeloblastosis virus transcriptase (Amersham Pharmacia Biotech, Upsalla) following standard protocols. A cDNA fragment corresponding to an AKT1-like K+ channel was amplified by PCR by using Taq polymerase (Amersham Pharmacia Biotech, Upsalla) and the degenerated sense 5'-TTYAAYTAYTTYTGGGTIMGITGYGC-3' and antisense 5'-GCYTCRTTYTGIARDATIACRTCYTC-3' primers designed from the conserved regions FNYFWVRCAK and EDVILQNEA present in AKT1 K+ channels from several plant species. The PCR product was cloned into the PCR2.1 vector by using the TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. The amino acid sequence deduced from the cDNA fragment showed the highest homology to the Arabidopsis AKT1.
Semi-quantitative PCR was performed from cDNA synthesized as described above. To check for the presence of CaHAK1 cDNA the sense 5'-ACTTCCTCATGATTGCTTGTG-3' and antisense 5'-CTGATTGAATTGACCGAGTTG-3'primers were used. To check for the presence of CaAKT1 cDNA the sense 5'-TCGGAGCCTCTATGGATGACT-3' and antisense 5'-GAAAATGTGAAACGCTTGAAC-3' primers were used, and for the presence of the elongation factor 1
cDNA, used as a control gene for its constitutive expression, the sense 5'-TTCACTGCCCAGGTCATCATC-3' and antisense 5'-TGTTCACTTCCCCTTCTTCTG-3' primers were used. The PCR reactions were run for 20, 22, 24, 26, 28, and 30 cycles. Semi-quantitative PCR by RT-PCR was repeated three times in all cases and a representative experiment is shown.
Nucleic acid gel-blot hybridizations
RNA gel-blot hybridizations were carried out by the northern technique as described elsewhere (Sambrook et al., 1989). Probe labelling, hybridization, and detection were performed by using the DIG high prime DNA labelling and detection starter kit II (Roche Biochemicals, Summerville, NJ) following the manufacturer's instructions. For RNA gel-blot hybridization, 30 µg of total RNA from roots were separated by electrophoresis in a formaldehyde–1.1% agarose gel and transferred to a nylon membrane. The CaHAK1 probe was synthesized from the full-length cDNA of CaHAK1 (Martínez-Cordero et al., 2004) and the CaAKT1 probe from the cDNA fragment isolated as described above.
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Results |
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Effect of K+ starvation on high-affinity K+ transport in pepper plants
Previous studies have shown that pepper plants induce a high-affinity K+ uptake system when they are deprived of K+ (Martínez-Cordero et al., 2004
) capable of depleting the external K+ concentration below 1 µM. The effect of the duration of K+ starvation on the development of the high-affinity K+ uptake component was studied. Plants were grown hydroponically for 1 month in one-fifth Hoagland solution and transferred to a K+-free nutrient solution for 2, 4, 6, 8, 24, 48, and 72 h. Control plants remained in the K+-containing solution. Then K+ depletion experiments from a 50 µM K+ external solution were performed. Control plants and plants starved of K+ for 2, 4, 6, or 8 h did not show K+ depletion (not shown). Plants starved of K+ for 24, 48, or 72 h were capable of depleting external K+ and the plots of the external K+ concentration versus time showed that the rates of K+ depletion increased as the time of K+ starvation of the plants increased. Plants starved of K+ for 72 h showed the highest rates of K+ depletion and were able to deplete external K+ from the 50 µM K+ solution to concentrations below 1 µM in 120 min (Fig. 1). The K+ depletion data for each plant starved of K+ for 72 h were fitted to a Michaelis–Menten equation and the Km and the Vmax values calculated. Twenty-one replicates were performed with one plant per replicate and an average Km of 6.2±0.5 µM K+ and Vmax of 1.72±0.24 µmol g–1 root DW min–1 were calculated.
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Localized induction of high-affinity K+ uptake
Experiments were designed to characterize the induction of the high-affinity K+ uptake further. By using a split root system, the effect of subjecting one-half of the root system to a K+-free solution and the other half to a K+-containing solution on the development of high-affinity K+ uptake was studied. In control experiments both halves of the root system were incubated in K+-containing or K+-free solutions. K+ depletion experiments were performed with each half of the root system in separate containers. The roots of plants incubated in the presence of K+ did not deplete external K+ and the roots of plants incubated in the K+-free solution showed the characteristic K+ depletion capacity of K+-starved plants described above (Fig. 2). The development of the high-affinity K+ uptake in the half of the root system incubated in the K+-free solution was independent of the presence or absence of K+ in the other half of the root system (Fig. 2). In parallel to the K+ depletion experiments, the internal K+ concentrations were determined in roots and shoots of the same plants that were used for the depletion experiments. The halves of the root system incubated in the absence of K+ showed lower concentrations than those incubated in the presence of K+. The K+ concentrations of the roots incubated in the absence of K+ were similar, independently of the presence or the absence of K+ in the other half of the root system and the same was true for the roots incubated in the presence of K+ (Fig. 3A). Therefore, the K+ concentration of one-half of the root was independent of the presence or absence of K+ in the solution where the other half of the root was incubated. The shoots of the plants with both halves of the root system incubated in the presence of K+ showed the highest concentrations of K+, followed by the shoots of plants with the roots split in K+-containing and K+-free solutions. Finally, the shoots of plants with both halves of the root incubated in the absence of K+ showed the lowest K+ concentrations (Fig. 3B).
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K+ concentrations of plant tissues in relation to high-affinity K+ uptake
The experiments with the split root system suggested that the K+ concentration of the roots determined the induction of the high-affinity K+ uptake. To relate high-affinity K+ uptake to the K+ concentration of roots and shoots, K+ depletion experiments were performed with plants grown under three different concentrations of K+, 1.4 mM K+, 0.7 mM K+, and 0.1 mM K+, in order to obtain plants with different K+ contents. After growing in these conditions for 1 month plants were transferred to a K+-free solution and K+ depletion experiments at days 0, 1, 2, and 3 after transferring the plants to the K+-free solution were carried out. Plants grown at 1.4 mM or 0.7 mM K+ showed similar K+ depletion capacities (Fig. 4A, B). At day 0 plants did not show K+ depletion. The rates of K+ depletion increased with increasing time of K+ starvation and on day 3 the plants depleted external K+ to values below 1 µM in 120 min. By contrast, the plants grown at 0.1 mM K+ performed differently. These plants showed a slight K+ depletion capacity on day 0 and on day 1 these plants showed a higher K+ depletion rate than the depletion rates of the plants grown at 1.4 or 0.7 mM K+. Plants grown in 0.1 mM K+ depleted external K+ to values below 1 µM in 120 min on day 2 and on day 3 these plants showed the highest rate of K+ depletion of the plants grown in 1.4, 0.7, or 0.1 mM K+, depleting external K+ to values below 1 µM before 120 min (Fig. 4C). In conclusion, plants grown at 0.1 mM K+ were able to reach higher rates of K+ depletion in a shorter time of K+ starvation. In parallel, the internal K+ concentrations were determined in roots and shoots of the same plants that were used for the K+ depletion experiments. The K+ concentration in the tissues decreased as the time of K+ starvation increased for all plants used. The K+ concentrations in the tissues of plants grown in 1.4 mM K+ or 0.7 mM K+ were similar for each day of K+ starvation and no significant differences were observed. Roots or shoots of plants grown in 0.1 mM K+ showed lower K+ concentrations than the roots or shoots of plants grown in 1.4 mM or 0.7 mM K+ for the corresponding days of K+ starvation (Fig. 5).
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NH4+, Cs+, and Na+ effects on high-affinity K+ transport
The sensitivity of the high-affinity K+ uptake of pepper plants to
Cs+, and Na+ was determined. Plants grown as described in the Materials and methods section and starved of K+ for 3 d were used for the experiments. The effects of Cs+, and Na+ on K+ uptake were studied by adding several concentrations of the cations to the solution containing 50 µM K+. The external concentrations of ranged from 10 µM to 10 mM, the Cs+ concentrations from 50 µM to 5 mM, and the Na+ concentrations from 5 mM to 200 mM. The rates of K+ depletion decreased as the concentrations of Na+, or Cs+ in the depletion solution increased. The data of K+ depletion in the presence of the inhibitors were fitted to Michaelis–Menten equations and the Km and Vmax values were calculated. The Vmax data in the presence of the different inhibitors were similar among them and similar to the Vmax in the absence of the inhibitors and no significant differences were observed (Table 1). Therefore it was concluded that Cs+, and Na+ competitively inhibited K+ uptake and the Ki values were calculated from several experiments with several concentrations of the inhibitors (Table 1). The average values of the Ki calculated from experiments with different concentrations of the inhibitors were (µM) 21.3±0.8 for 31.1±0.6 for Cs+, and (mM) 6.2±1.7 for Na+.
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Effect of NH4+ in the growth solution on high-affinity K+ uptake
As described above, the presence of
in the external solution strongly inhibited high-affinity K+ uptake of plants grown in the absence of . Experiments were carried out to determine the effect of the presence of in the solution used to grow the plants on the sensitivity of high-affinity K+ transport to . Plants were grown in the one-fifth Hoagland solution with 
as the nitrogen source (2.8 mM ) or in a one-fifth Hoagland solution with 50% of the nitrogen supplied as and 50% as (1.4 mM NH4NO3). After growing the plants for 1 month, they were subjected to K+ starvation by transferring the plants for 3 d to solutions containing the same nutrients but deprived of K+. Control plants remained in the K+-containing solutions. Depletion experiments were performed from solutions containing 50 µM K+ and the effect of was studied by adding 1 mM . Control experiments were performed in the absence of . Plants that were not starved of K+ did not show K+ depletion (not shown). Plants grown in the absence of (2.8 mM ) depleted external K+ to values below 1 µM K+ in the absence of and the presence of 1 mM in the solution used for the depletion experiment almost abolished the K+ depletion capacity. Plants grown in the presence of (1.4 mM NH4NO3) were able to deplete external K+ in the absence of as well as in the presence of 1 mM in the external solution. In the absence of in the depletion solution these plants depleted external K+ to values around 1 µM, whereas in the presence of 1 mM the plants depleted external K+ to concentrations values around 10 µM (Fig. 6).
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In parallel with the K+ depletion experiments, the internal K+ concentrations were determined in the roots of the same plants that were used for the depletion experiments. K+-starved plants showed lower K+ concentrations than non-starved plants, and the roots of K+-starved plants grown in the presence of 1.4 mM NH4NO3 showed a slightly lower K+ concentration than the roots of K+-starved plants grown without
(Fig. 7).
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Expression of CaHAK1
A high-affinity K+ transporter of the HAK family, CaHAK1, has been identified in roots of pepper plants (Martínez-Cordero et al., 2004
). CaHAK1 expression was studied by northern analyses with RNA isolated from roots of the plants used for the K+ depletion experiments described above. In the experiments with the split root system, CaHAK1 expression could not be detected in the halves of the root system incubated in the presence of K+ whereas CaHAK1 expression was detected in the halves of the root system incubated in the absence of K+, independently of the presence or absence of K+ in the solution where the other halves of the root system were incubated (Fig. 8A).
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In the experiments where the effect of the presence of
in the growth solution was studied, it was observed that CaHAK1 expression was not detected in the roots of plants grown in the presence of K+ and that it was strongly induced in the roots of the plants grown in the absence of and starved of K+ for 3 d. The presence of in the growth solution reduced the expression of CaHAK1 (Fig. 8B).
Expresssion of CaAKT1
In Arabidopsis, the K+ channel AKT1 mediates the -insensitive component of the K+ uptake in the high-affinity range of K+ concentrations (Hirsch et al., 1998; Spalding et al., 1999). A cDNA fragment of the AKT1 homologue of pepper, CaAKT1, was cloned here to study if the induction of the -insensitive K+ uptake shown by pepper plants was related to an increase in CaAKT1 expression. Northern analyses did not detect messenger RNA of CaAKT1 and semi-quantitative PCR was performed as an alternative method. As a control, CaHAK1 expression was also determined. RNA was reversed transcribed and then cDNA fragments corresponding to CaHAK1, CaAKT1, and the elongation factor 1 were amplified by PCR in several reactions for different cycles. After 20 cycles of amplification, the CaHAK1 cDNA was only detected in K+-starved plants. The amount of CaHAK1 cDNA was higher in plants grown in the absence of than in its presence (Fig. 9), in agreement with the results obtained by the northern analysis. CaAKT1 cDNA was not detected in any sample (Fig. 9) and the amount of the elongation factor 1 cDNA was the same in all samples as expected for a constitutive gene (Fig. 9). Increasing the PCR cycles showed that CaAKT1 cDNA could be detected after 30 cycles of amplification and that similar amounts of cDNA were observed in all samples (Fig. 9). Under these conditions, CaHAK1 cDNA was detected in all samples with the highest amount for plants starved of K+ and grown in the absence of and the elongation factor 1 cDNA was the same in all samples (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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It is shown here that pepper plants grown in the absence of
show a high-affinity K+ uptake system capable of depleting K+ in external solutions to values below 1 µM. This system is locally induced in the root zone subjected to K+ starvation. The rates of K+ uptake increased as the time of K+ starvation increased and the internal root K+ concentration decreased. In those plants grown in the absence of the system is inhibited by the presence of Cs+, and Na+. When plants are grown in the presence of an -insensitive component comes into play. The expression studies by northern and semi-quantitative PCR with CaHAK1 and CaAKT1 suggest that members of group I of the KT/HAK/KUP family may preferentially participate in the -sensititve component of the high-affinity K+ uptake, and AKT1-like channels in the -insensitive component.
A rapid high-affinity K+ uptake which depleted external K+ to concentrations below 1 µM in 120 min was induced in pepper plants after 3 d of K+ starvation (Fig. 1). An allosteric model has been suggested in barley for the regulation of the induction of high-affinity K+ uptake by internal K+ (Glass, 1976). A similar model may also apply to the K+ transport system in pepper. The signal triggering the induction of the high-affinity K+ uptake should be originated in the root exposed to the solution without K+. In the experiments with the split root system, only the half of the root exposed to the K+-free solution showed high-affinity K+ uptake, and this was independent of the presence or the absence of K+ in the solution bathing the other half of the root system (Fig. 2). In addition, the roots exposed to the K+-free solution showed lower K+ concentrations than the roots exposed to the K+-containing solution, also independently of the presence or absence of K+ in the solution bathing the other half of the root system (Fig. 3A). The participation of the shoot in the generation of a systemic signal to induce the high-affinity K+ uptake in roots is unlikely. The plants with the root system split in K+-free and K+-containing solutions had the same shoot and only the half of the root in the K+-free solution showed high-affinity K+ uptake. However, the participation of the shoot cannot be excluded. The K+ concentration of the shoots of the plants with the root split in K+-containing and K+-free solution was intermediate between that of the plants with both halves in K+-containing solution and that of the plants with both halves in K+-free solution. Therefore, it is possible that if the shoot K+ concentrations are further decreased, the roots develop high-affinity K+ uptake even if the roots are bathed in K+-containing solution. In the acquisition of other nutrients such as iron, the regulation of the system for high-affinity uptake involves both local and systemic signals (Vert et al., 2003).
The results described above indicated that, in the experimental conditions used here, the signal inducing the high-affinity K+ uptake originated in the root exposed to the K+-free solution. However, two possibilities existed, either the root responded to the lack of K+ in the external solution or to the decrease in the internal K+ concentration. Pepper plants did not develop a rapid high-affinity K+ uptake until they had been incubated in solutions without K+ for 3 d (Fig. 1). This suggested that the decrease in the internal K+ concentration and not the absence of external K+ was the signal to induce the K+ transport system and experiments were designed to address this point. Plants were grown at three different K+ concentrations and assayed for high-affinity K+ uptake after different times of K+ starvation (Fig. 4). In parallel, the internal K+ concentrations in roots and shoots were determined (Fig. 5). Roots with similar K+ concentrations showed similar rates of K+ depletion and, for example, the roots grown at 1.4 mM or 0.7 mM K+ and starved for K+ for 2 d showed similar concentrations of K+ in their roots and similar rates of K+ depletion to the plants grown in the presence of 0.1 mM K+ (0 d of K+ starvation) (Figs 4, 5A). In addition, plants starved of K+ for short periods of time (2, 4, 6, or 8 h) showed similar internal K+ concentrations to control plants and did not deplete external K+. The results showed that when the root K+ concentration decreased to a concentration below 30 mg g–1 root DW, a rapid, high-affinity K+ uptake capable of depleting external K+ in 120 min was observed (Figs 4, 5). That threshold was reached sooner during K+ starvation for the plants grown at the lowest K+ concentration (0.1 mM K+). In agreement with this, in the experiments described above with the spilt root system, only the roots with K+ concentrations lower than the 30 mg g–1 root DW threshold showed a high-affinity K+ uptake (Figs 2, 3).
Cs+, and Na+ competitively inhibited the high-affinity K+ uptake shown by pepper plants grown in the absence of (Table 1). and Cs+ inhibited in the micromolar range of concentrations and Na+ in the millimolar range. These results strongly suggested that a transporter of the HAK family was mediating the K+ influx. HAK transporters are inhibited by and Na+ and have been shown to transport Cs+ and Na+ (Santa-María et al., 1997; Rubio et al., 2000; Banuelos et al., 2002). In agreement with this, pepper plants starved of K+ also transported Cs+ with high-affinity (not shown).
is known to cause toxicity in plants, especially at low K+ concentrations (Rufty et al., 1982). Interestingly, the sensitivity of high-affinity K+ uptake to depended on the conditions of plant growth. Thus, plants grown in the absence of showed a high-affinity K+ uptake highly sensitive to whereas plants grown in the presence of showed a high-affinity K+ uptake which was almost completely insensitive to (Fig. 6). Plants grown in the presence of and assayed for K+ depletion in the absence of depleted external K+ to the same values below 1 µM K+ as plants grown in the absence of . However, plants grown in the presence of and assayed for K+ depletion in the presence of 1 mM depleted external K+ to higher values, around 10 µM K+. This could indicate that in plants grown in the presence of high-affinity K+ uptake was mediated by two different systems, one sensitive to and the other one insensitive to . In the presence of in the depletion experiment the -sensitive component is abolished and K+ is taken up through the -insentive component which fails to take up K+ from external K+ concentrations lower than 10 µM. A possible explanation is that the -sensitive component is mediated by CaHAK1, which is probably a K+-H+ symporter, as suggested for HAK transporters of group I of other species (Rodríguez-Navarro, 2000; Very and Sentenac, 2003), and the -insensitive component by a K+ channel. According to this hypothesis, at given values of the membrane potential and internal K+ concentrations, and in the absence of in the depletion experiment, CaHAK1 would mediate K+ uptake at external K+ concentrations lower than 10 µM where the channel fails. In the presence of CaHAK1 would be inhibited and the channel would not be able to take up K+ from external K+ concentrations lower than 10 µM. It is worth mentioning that the roots of plants grown in the presence or absence of showed similar concentrations of K+ (Fig. 7). -sensitive and -insensitive components of high-affinity K+ uptake have been described in Arabidopsis and barley (Spalding et al., 1999; Santa-María et al., 2000). In Arabidopsis, the -insensitive component is mediated by the K+ channel AKT1 which contributes to K+ uptake between 55% and 63% from solutions containing between 10 and 1000 µM in the absence of (Spalding et al., 1999). In pepper the contribution of the -insensitive component in plants grown in the absence of seems to be much less important than in Arabidopsis, because, in pepper, almost completely inhibited the high-affinity K+ uptake. In barley, as in pepper, -sensitive and -insensitive components of high-affinity K+ uptake have been described in plants grown in the absence or presence of respectively (Santa-María et al., 2000). It can be speculated that, in different plant species, the -sensitive and -insensitive pathways contribute differently to high-affinity K+ uptake.
The results on the expression of the gene encoding CaHAK1 support the hypothesis that a transporter of the KT/HAK/KUP family is mediating the -sensitive high-affinity K+ uptake shown in the roots starved of K+. CaHAK1 was only induced in the half of the root system exposed to the K+-free solution regardless of the presence or absence of K+ in the other half of the root system (Fig. 8A). This lends support to the notion that the induction of the high-affinity K+ uptake is a localized process that does not travel within the plant. A member of group I of the KT/HAK/KUP family of K+ transporters may preferentially participate in high-affinity K+ uptake in plants grown in the absence of as suggested by the expression pattern of CaHAK1. The expression of CaHAK1 was induced in K+-starved plants and the induction was higher in plants grown in the absence of than in the presence of (Fig. 8B). It is worth mentioning that although the roots of plants grown in the presence of showed lower K+ concentrations than those of plants grown in the absence of it (Fig. 7), they did not show higher expression levels of CaHAK1 (Fig. 8). When plants are grown in the presence of an additional system which is not inhibited by contributes to high-affinity K+ uptake. The results obtained by semi-quantitative PCR (Fig. 9) showed that the pepper AKT1 is expressed in plants grown in the presence of and starved of K+. It is therefore possible that an AKT1-like channel participates in the -insensitive K+ uptake in the micromolar range of K+ concentrations shown by pepper plants grown in the presence of . CaAKT1 is also expressed in K+-grown plants and in plants grown in the absence of and starved of K+, suggesting that as described for AtAKT1 (Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), CaAKT1 is a constitutively expressed gene. Thus, if CaHAK1 is dominating the high-affinity K+ uptake in plants grown in the absence of the CaAKT1 function should be subjected to post-transcriptional regulation.
Finally, the high-affinity K+ uptake system of pepper has been shown to be inhibited by millimolar concentrations of Na+. Maintaining high K+/Na+ ratios in the plant tissues is crucial for salt tolerance and the selectivity of the high-affinity K+ transporters may play a role in the tolerance to Na+ (Niu et al., 1995). Pepper is a salt-sensitive crop (Maas and Hoffman, 1977) and increasing the selectivity for K+ against Na+ could lead to obtaining plants more tolerant to salinity, and high-affinity K+ transporters as CaHAK1 may be determinants for salt tolerance.
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This work was supported by grant PB/62/FS702 from Consejería de Ciencia, Tecnología, Industria y Comercio de la Región de Murcia (Programa Séneca).
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