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Journal of Plant Physiology 167 (2010) 1494–1499

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .de / jp lph

ystems involved in K+ uptake from diluted solutions in pepper plants asevealed by the use of specific inhibitors

rancisco Rubioa,∗ , Laura Arévaloa, Fernando Caballeroa, María Angeles Botellab, José Salvador Rubioa,rancisco García-Sáncheza, Vicente Martíneza

Departamento de Nutrición, Centro de Edafología y Biología Aplicada del Segura-CSIC, Campus de Espinardo, 30100 Murcia, SpainDepartamento de Biología Aplicada, Universidad Miguel Hernández, Carretera de Beniel Km 3.2, 03312 Orihuela Alicante, Spain

r t i c l e i n f o

rticle history:eceived 11 February 2010eceived in revised form 13 May 2010ccepted 27 May 2010

eywords:otassiumigh-affinitybsorption

a b s t r a c t

Here, the contribution of the HAK1 transporter, the AKT1 channel and a putative AtCHX13 homologto K+ uptake in the high-affinity range of concentrations in pepper plants was examined. The limiteddevelopment of molecular tools in pepper plants precluded a reverse genetics study in this species.By contrast, in the model plant Arabidopsis thaliana, these type of studies have shown that NH4

+ andBa2+ may be used as specific inhibitors of the two K+ uptake systems to dissect their contribution inspecies in which, as in pepper, specific mutant lines are not available. By using these inhibitors togetherwith Na+ and Cs+, the relative contributions of CaHAK1, CaAKT1 and a putative AtCHX13 homolog toK+ acquisition from diluted solutions under different regimens of K+ supply were studied. The results

+

epper showed that, in plants completely starved of K , the gene encoding CaHAK1 was highly expressed and

this system is a major contributor to K+ uptake. However, K+ concentrations as low as 50 �M reducedCaHAK1 expression and the CaAKT1 channel came into play, participating together with CaHAK1 in K+

absorption. The contribution of a putative AtCHX13 homolog seemed to be low under this low K+ supply,but it cannot be ruled out that at higher K+ concentrations this system participates in K+ uptake. Studiesof this type allow extension of the tools developed in model plants to understand nutrition in important

crops.

ntroduction

Potassium is an essential macronutrient for plants that com-oses up to 10% of the total dry weight. It fulfills important functionsnd its concentration is kept constant in the cytosol at around00 mM. This is in contrast with the wide range of K+ concentra-ions that plant roots encounter in the soil solution. To acquire K+,he plasma membrane of root cells is furnished with selective high-nd low-affinity K+ transporters that ensure K+ supply from manyifferent K+ concentrations. At low K+ concentrations in the soilolution, which are found in many agricultural soils, the functionf systems that contribute to high-affinity K+ uptake is crucial toustain plant growth and productivity (Clarkson, 1985). In someases, the presence of other ions such as Na+ or NH4

+ may interfereith K+ uptake and impair plant growth, especially at low external

+ concentrations (Flowers and Läuchli, 1983; Rufty et al., 1982).The first high-affinity K+ uptake system to be kinetically charac-

erized was from barley (Hordeum vulgare), and it showed a Km for+ of 18 �M, and no discrimination between K+ and Rb+ and low-

∗ Corresponding author. Tel.: +34 968396351; fax: +34 968396213.E-mail address: frubio@cebas.csic.es (F. Rubio).

176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.jplph.2010.05.022

© 2010 Elsevier GmbH. All rights reserved.

affinity for Na+ (Epstein et al., 1963). Similar systems have beendescribed in other plant species (Epstein, 1973; Kochian and Lucas,1982; Maathuis and Sanders, 1994). Further studies on the regula-tion of K+ absorption into barley roots showed that there is a rapidup-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 thelow-affinity range of concentrations is mediated by K+ channels(Maathuis and Sanders, 1996). Molecular approaches have led tothe identification, in several plant species, of families of genes thatencode K+ transporters and K+ channels (Rodríguez-Navarro, 2000;Very and Sentenac, 2003). Members of the KT/HAK/KUP family ofK+ transporters may be involved in high-affinity K+ uptake intothe roots. By heterologous expression in yeast, it has been shownthat HAK1-type transporters of several plant species show char-acteristics that are in agreement with the high-affinity K+ uptakeobserved in the corresponding plant roots (Banuelos et al., 2002;

Santa-María et al., 1997; Martínez-Cordero et al., 2004; Nieves-Cordones et al., 2008). They mediate Rb+ uptake with Km valuesfor Rb+ between 20 and 1 �M, no discrimination between Rb+ andK+ and the genes encoding these transporters are induced in theroots of K+-starved plants in parallel with development of high-

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F. Rubio et al. / Journal of Plan

ffinity K+ uptake. Therefore, these transporters are probably majorontributors to high-affinity K+ uptake in the plant species stud-ed. In Arabidopsis (Arabidopsis thaliana), the K+ transporter HAK5xpressed in yeast also shows high-affinity for Rb+ (12.6 �M), noiscrimination between K+ and Rb+, and inhibition by Na+ and Cs+

Rubio et al., 2000). The gene encoding it is induced in the roots of+-starved plants (Armengaud et al., 2004; Shin and Schachtman,004). On the other hand, the Arabidopsis K+ channel AKT1 has beenhown to mediate low-affinity K+ uptake (Gierth et al., 2005), whichs strongly blocked by Ba2+ and Cs+ and almost unaffected by Na+

Bertl et al., 1997). The gene encoding AtAKT1 is expressed con-titutively (Lagarde et al., 1996). Importantly, the AtAKT1 channelas been described as an important component of the K+ uptakepparatus, even in the high-affinity range of K+ concentrationsHirsch et al., 1998; Spalding et al., 1999). Studies on Arabidop-is that combined the use of T-DNA insertion lines in AtHAK5 andtAKT1 and specific inhibitors revealed the relative contributionf each of these systems to K+ uptake from low concentrationsRubio et al., 2008). AtHAK5 mediates NH4

+-sensitive high-affinity+ uptake of high concentrative capacity, whereas AtAKT1 mediatesa2+-sensitivity K+ uptake of a much lower concentration capac-

ty. These two systems are the only ones involved in K+ uptakehen external K+ is below 50 �M (Rubio et al., 2010). In bar-

ey, NH4+-sensitive high-affinity K+ uptake, probably mediated by

vHAK1, and Ba2+-sensitive high-affinity K+ uptake, have also beenescribed (Santa-María et al., 2000). At K+ concentrations higherhan 50 �M, members of the cation:proton antiporter family suchs AtCHX13 may contribute to K+ uptake (Zhao et al., 2008).

The studies performed in model plant species such as Ara-idopsis have provided essential and valuable information onigh-affinity K+ uptake in plants. These studies have shown thatH4

+ or Ba2+ may be used in plant species in which specific mutantsre not available as tools to dissect components of K+ uptake fromiluted solutions (Rubio et al., 2008). These tools enable the studyf the relative contribution of the HAK transporter, the AKT chan-el, or other systems. This is case for pepper, a globally importantrop in which information on the entities mediating K+ uptake iscarce. Recently, cDNAs of the corresponding HAK1 transporter andhe AKT1 channel have been isolated from pepper plants. CaHAK1s up-regulated by K+ starvation and encodes a high-affinity K+

ransporter, whereas CaAKT1 is expressed constitutively (Martínez-ordero et al., 2004, 2005). Specific mutants affecting these twoenes in pepper are lacking, and the difficulties in transformingepper plants preclude advancing our knowledge of K+ nutrition

n this species. Here, we used NH4+, Ba2+, Na+ and Cs+ to study the

elative contributions of CaHAK1 and CaAKT1 and other systemso K+ uptake from diluted solutions. In addition, long-term exper-ments were carried out to characterize growth of pepper plants

hen K+ was supplied at concentrations within the operation ofigh-affinity K+ uptake.

aterials and methods

lant growth

Seeds of pepper (Capsicum annum L., cv. requena) were pre-ydrated with an aerated 0.5 mM CaSO4 solution for 72 h anderminated in vermiculite at 28 ◦C. After 2 d, the seedlings werelaced in 15 L containers filled with modified one-fifth Hoaglandı̌solution, which consisted of the following macronutrients (mM):

.4 KCl, 1.4 Ca(NO3)2, 0.1 Ca(H2PO4)2, 0.35 MgSO4 and the follow-

ng micronutrients (�M): 50 CaCl2, 12.5 H3B03, 1 MnSO4, 1 ZnSO4,.5 CuSO4, 0.1 H2MoO4, and 10 Fe-EDDHA. Plants were subjectedo K+ starvation by growing the plants in the same solution withoutCl for 3 d. Plants were grown in a controlled-environment cham-

ology 167 (2010) 1494–1499 1495

ber with a 16 h light–8 h dark cycle and air temperatures of 25 and20 ◦C, respectively. The relative humidity was 65% (day) and 80%(night) and the photon flux density 550 �mol m−2 s−1. Modest aer-ation was provided. The nutrient solution was replaced with freshsolution weekly.

K+ depletion experiments

10-d-old plants grown as described above were employed for K+

depletion experiments. For K+ starvation, plants were transferredfor 3 d to the nutrient solution deprived of K+ described above.Plants were rinsed in a cold K+-free nutrient solution and trans-ferred at time zero to 250 mL containers with a K+-free nutrientsolution supplemented with 50 �M KCl and NH4Cl, BaCl2, NaCl orCsCl, as indicated. Samples of 1 mL were taken at intervals for 6 hand their K+ concentrations determined by atomic emission in aPerkin-Elmer (Boston, MA) AAnalyst 400 spectrophotometer. Therates of K+ depletion were calculated from the decrease in K+ dur-ing the lineal phase of the depletion curve and referred to the dryweight of the root. Average and standard error values are reported.At the end of the experiment, plants were separated into roots andshoots and the fresh weight determined. Then, roots and shootswere dried at 65 ◦C for 4 d and the dry weight determined.

Plant growth, internal K+ contents, K+ net uptake experiments andpercentage K+ transported to the shoot

10-d-old plants grown as indicated above were starved of K+ for3 d and then transferred to a solution containing 50 �M K+ for 14d. 1 mM NH4

+, 1 mM Ba2+, 10 mM Na+ or 1 mM Cs+ were added asindicated. Plant material was harvested at days 0, 7 and 14 aftertransferring the plants to the 50 �M K+ solution. Plants were sepa-rated into roots and shoots and the fresh weight determined. Then,roots and shoots were dried at 65 ◦C for 4 d and the dry weightdetermined. To study growth, plant dry weight was plotted againsttime. Chemical analyses of plant material were carried out afterdigestion with HNO3–HClO4 (2:1). K+ concentrations were deter-mined by atomic absorption spectrometry. The rates of net uptakeof K+ were calculated between two different harvests from theinternal K+ contents in plants and the root dry weights. For compar-ison, the rates of K+ uptake are compared to the control without theinhibitors, which is taken as 100%. The percentage of K+ transportedto the shoot was calculated between two different harvests fromthe increase in K+ content in the shoot referred to the increase intotal K+. Average values are reported and error bars denote standarderrors.

Nucleic acid gel blot hybridizations

RNA gel blot hybridizations were carried out by the RNAtechnique as described elsewhere (Sambrook et al., 1989). Probelabeling, hybridization and detection were performed by usingthe DIG high prime DNA labeling and detection starter kit II(Roche Biochemicals, Summerville, NJ) following the manufac-turer’s instructions. For RNA gel blot hybridization, 30 �g oftotal RNA from roots were separated by electrophoresis in aformaldehyde–1.1% agarose gel and transferred to a nylon mem-brane. The probe was synthesized from the full-length cDNA ofCaHAK1 (Martínez-Cordero et al., 2004).

Results

K+ uptake from diluted solutions in K+-starved plants

Pepper plants starved of K+ have been previously shownto develop NH4

+-sensitive high-affinity K+ uptake. These plants

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496 F. Rubio et al. / Journal of Plan

epleted external K+ in 120 min from a 50 �M K+ solutionMartínez-Cordero et al., 2004, 2005). Here examined K+ uptaken the range of operation of the high-affinity system and its sensi-ivity to NH4

+, Ba2+, Na+ and Cs+. Plants were grown in a modifiedoagland solution containing 1.4 mM K+ for 5 d and then starvedf K+ by transferring the plants to a solution with no K+ added ford. Then plants were used for K+ depletion experiments in solu-

ions that contained 50 �M K+. In some cases NH4+, Ba2+, Na+ or

s+ were added. Plants completely depleted external K+ from the0 �M solution in less than 150 min in the absence of inhibitors. Theddition of Ba2+ or Na+ had little, effect but the addition of NH4

+

educed the rate of K+ depletion and plants were unable to deplete+ from the solutions that contained NH4

+. The addition of Cs+ alsoeduced K+ uptake, producing stronger inhibition than NH4

+.To quantify K+ uptake and the effects of the inhibitors, the rates

f K+ uptake per unit of root dry weight were calculated from theinear phase of the curves in the depletion experiments. We foundhat the addition of Ba2+ or Na+ did not affect the rates of K+ uptake,hereas NH4

+ reduced a 35% and Cs+ a 72% K+ uptake. The additionf Ba2+ and NH4

+ together reduced the rates to the same extent asH4

+ alone. Within the solutions that contained Ba2+ plus NH4+,

he addition of Na+ did not produce any effect and the addition ofs+ reduced K+ uptake rates to the same level as Cs+ alone.

ffect of the presence of K+ during growth on K+ uptake fromiluted solutions

Growth conditions, including the presence of K+, may affect theontribution of the different systems involved in K+ uptake in theigh-affinity range of concentrations. To further study the contri-ution of those systems in pepper plants growing under steadytate conditions with a 50 �M K+ supply, a long-term experimentas designed. Plants were grown for 5 d in the modified Hoagland

olution with 1.4 mM K+, starved of K+ for 3 d and then transferredo a solution with 50 �M K+, in the absence or in the presence of thenhibitors for 14 d. Plant organs were harvested at different timeoints to determine their dry weight, K+ contents and K+ concen-rations.

The presence of 1 mM NH4+, 1 mM Ba2+ or 10 mM Na+ alone did

ot affect plant growth. The presence of 1 mM NH4+ + 1 mM Ba2+

lightly reduced plant growth, and at d 21, the biomass of plantsrowing with these two inhibitors was significantly lower (Fig. 2).he presence of 10 mM Na+ in the NH4

+ + Ba2+-containing solutionsroduced an important reduction of plant growth. Finally, the pres-nce of 1 mM Cs+ strongly reduced plant growth, irrespective of theresence of other inhibitors in the growth solution.

K+ concentrations of roots and shoots were determined after 7of plant growth in the presence of 50 �M K+ under the different

nhibitor treatments. All inhibitors reduced root and shoot K+ con-entrations, with the exception of Na+, which did not produce anyignificant change in shoot K+ concentrations (Fig. 3). The pres-nce of 1 mM Cs+ produced the most significant reduction in K+

oncentrations. Interestingly, the presence of 1 mM Ba2+ or 10 mMa+ produced reductions of root K+ concentrations similar to thoseith 1 mM NH4

+. Ba2+ led to larger reductions in shoot K+ concen-rations, whereas Na+ did not affect shoot K+ concentrations. Theddition of NH4

+ and Ba2+ together reduced K+ concentrations tohe same level of Cs+ alone and to a larger extent than when thesewo inhibitors were added separately. The addition of Na+, Cs+ oroth to the solutions containing NH4

+ + Ba2+ did not further reduce+ concentrations.

Based on the increase of plant K+ contents, the rates of K+ absorp-ion were calculated after the 7 d period of growth in the presencef 50 �M K. To compare the effect of the inhibitors, the rates of K+

ptake in the different treatments were calculated as a percent-ge of the rate in the absence of inhibitors (Fig. 4). We found that

iology 167 (2010) 1494–1499

the presence of 1 mM NH4+ reduced the rate of K+ uptake 20%, the

presence of 1 mM Ba2+ 60%, the presence of 10 mM Na+ did notproduce any effect, and the presence of 1 mM Cs+ produced a reduc-tion of 92% on the rate of K+ uptake. The presence of both 1 mMNH4

+ + 1 mM Ba2+ reduced K+ uptake 81%. The addition of Na+ tothe NH4

+ + Ba2+-containing solution did not produce any effect. Bycontrast, the addition of Cs+ further decreased K+ uptake.

The percentage of K+ that was transported to the shoot was alsocalculated for plants subjected to the different treatments (Fig. 5).The presence of NH4

+ or Na+ did not affect the percentage of K+

transported to the shoot, the presence of Ba2+ reduced it, and thepresence of Cs+ produced a further reduction. The presence of NH4

+

and Ba2+ together reduced the percentage of K+ transported to alarger extent than the presence of these inhibitors separately andto the same level than Cs+ alone. The addition of Na+ or Cs+ to theNH4

+ + Ba2+-containing solutions did not further affect K+ trans-port.

CaHAK1 expression

CaHAK1 expression has been shown to be up-regulated by K+

starvation. Thus, to further study the contribution of CaHAK1 to K+

uptake in plants growing with 50 �M K+, its expression was stud-ied under that K+ supply and under complete K+ starvation as acontrol. Plants were grown 10 d in the growth solution contain-ing 1.4 mM K+ and then transferred for 14 d to a K+-free solutionor to a solution containing 50 �M K+. Plants grown for 14 d in theabsence of K+ showed high levels of CaHAK1 transcripts (Fig. 6), asdescribed previously. By contrast, in plants grown in solutions thatcontained 50 �M K+, the levels of CaHAK1 mRNA greatly decreasedand CaHAK1 messenger was barely detected.

Discussion

Pepper is an important crop world-wide and, as in other plantspecies, its productivity greatly depends on proper crop man-agement, including K+ fertilization. One approach to improve K+

capture efficiency of crops may be based on the identification andcharacterization of the systems involved in K+ acquisition. Candi-dates for these systems are members of group I of the KT-HAK-KUPfamily, AKT1 channels (Rodríguez-Navarro and Rubio, 2006), class 2HKT transporters (Platten et al., 2006; Horie and Schroeder, 2004)and members of the cation:proton antiporter family (Zhao et al.,2008). The contribution of these systems may differ among speciesor may be differentially affected by the growth conditions, depend-ing on the species. For example, in tomato an NH4

+-sensitiveK+ uptake system, probably mediated by the corresponding HAKtransporter LeHAK5, has been suggested to dominate high-affinityK+ uptake both in plants grown with or without NH4

+ (Nieves-Cordones et al., 2007). By contrast, in Arabidopsis (Rubio et al., 2008)or pepper plants (Martínez-Cordero et al., 2004, 2005), the NH4

+-sensitive component dominates in plants grown without NH4

+

whereas in NH4+-grown plants, an NH4

+-insensitive component isthe major contributor to K+ uptake. HKT-mediated high-affinity K+

uptake has been described mainly in monocot representatives, butin dicots, such as pepper, these transporters are probably involvedin Na+ removal from the xylem (Horie and Schroeder, 2004). TheArabidopsis AtCHX13 K+ transporter in yeast and plant cells showsa Km for K+ uptake of 136 and 196 �M, respectively, and it has beensuggested to promote K+ uptake in plants when K+ is limiting in the

environment (Zhao et al., 2008).

The use of Arabidopsis mutant lines and specific inhibitors of HAKtransporters and AKT channels has allowed the demonstration that,in Arabidopsis, the HAK transporter mediates the NH4

+-sensitiveand the AKT1 channel the NH4

+-insensitive, Ba2+-sensitive com-

F. Rubio et al. / Journal of Plant Physiology 167 (2010) 1494–1499 1497

Fig. 1. Rates of K+ depletion in K+-starved plants and effects of inhibitors. Pepperplants were starved of K+ for 3 d and then transferred to solutions containing 50 �MK+ in the absence or in the presence of 1 mM NH4

+, 1 mM Ba2+, 10 mM Na+ or 1 mMCs+ as indicated. At different time points samples of external solution were takenand their K+ concentration determined. Rates of K+ depletion were calculated for thelDl

po5t2upCbot2

a(aweuirrtCgccibce

aohsB1dti5st

Fig. 2. Growth of plants in the presence of 50 �M K+ and inhibitors. Plants starved

Here, it is shown that the gene is repressed by growing the plants

inear phase of the depletion curve a referred to the corresponding root dry weight.ata are averages of 5 repetitions and error bars denote standard error. Different

etters indicate significant differences (p < 0.05) according to Tukey test.

onent of K+ uptake, respectively (Rubio et al., 2008), and that nother system participates in K+ uptake when external K+ is below0 �M (Rubio et al., 2010). At higher K+ concentrations, other sys-ems, such as AtCHX13, may contribute to K+ uptake (Zhao et al.,008). In yeast cells, AtCHX13 mediates Cs+- and Na+-sensitive K+

ptake. The presence of 10 mM Cs+ or 10 mM Na+ almost com-letely inhibits K+ uptake, showing Na+ stronger inhibition thans+. On the other hand, micromolar concentrations of Cs+ stronglylock AtAKT1, and millimolar concentrations of Na+ have no effectn this channel (Bertl et al., 1997). Finally, micromolar concentra-ions of Cs+ and millimolar of Na+ inhibit AtHAK5 (Rubio et al.,000).

In pepper plants, a high-affinity K+ transporter, CaHAK1, andn inward-rectifier K+ channel CaAKT1 have been identifiedMartínez-Cordero et al., 2004, 2005). Because CaHAK1, CaAKT1nd a putative pepper homolog of AtCHX13 may mediate K+ uptakeith different concentrative capacities and different responses to

nvironmental conditions, determining their contribution to K+

ptake in crops such as pepper may provide information that willmprove K+ capture efficiency. However, in pepper plants, the cor-esponding mutant lines are not available, and obtaining them mayequire transgenic approaches which are not straightforward inhis species. Therefore, here we used inhibitors such as NH4

+, Ba2+,s+ or Na+, which differentially affect these transport systems, toain information about their contributions to K+ uptake from lowoncentrations. These studies were performed at an external K+

oncentration of 50 �M with K+-starved plants, where K+ net fluxs dominated by K+ influx (Britto and Kronzucker, 2006). It shoulde noted that, if experimental conditions make K+ efflux a relevantomponent of K+ net flux, differential effects of the inhibitors on K+

fflux systems should be also considered.As described previously (Martínez-Cordero et al., 2004, 2005)

nd here (Fig. 1) pepper plants grown without NH4+ and starved

f K+ for 3 d developed a high-affinity K+ uptake system that isighly sensitive to NH4

+ and Cs+ and insensitive to Na+. Here wehow that it is also insensitive to Ba2+ (Fig. 1). The insensitivity toa2+ and to Na+ suggest a low contribution of CaAKT1 (Bertl et al.,997) or an AtCHX13 homolog (Zhao et al., 2008) to K+ uptake fromiluted solutions, respectively. This system shows a great concen-rative capacity, as these plants completely depleted external K+

n less than 300 min (not shown) at external K+ concentrations of0 �M. At the same time, these plants show high levels of expres-ion of CaHAK1 (Fig. 6). Collectively, these results strongly suggesthat CaHAK1 is a major contributor to K+ uptake in these plants. It

of K+ for 3 d were transferred to a solution with 50 �M K+ in the absence of inthe presence of 1 mM NH4

+, 1 mM Ba2+, 10 mM Na+ or 1 mM Cs+ as indicated. Atdifferent time points plants were harvested and their dry weight determined. Dataare averages of 5 repetitions and error bars denote standard error.

is possible that other members of the KT-HAK-KUP family, insen-sitive to NH4

+, also contribute in this K+ uptake. For example, inbarley, NH4

+ represses HvHAK1b but not its close relative HvHAK1(Fulgenzi et al., 2008). This is an attractive hypothesis that needs tobe further studied.

A different picture was observed when plants were grown in thepresence of 50 �M K+. Under these conditions, plants still devel-oped K+ uptake, but it was less sensitive to NH4

+ and much moresensitive to Ba2+ and Cs+ (Fig. 4). In addition, these plants showedlow levels of expression of CaHAK1 (Fig. 6). These results suggestthat, in these plants grown with K+, CaHAK1 contributes less thanin plants grown with no K+ for 3 d. Under these conditions, a newsystem sensitive to Ba2+ and Cs+ and insensitive to Na+ comes intooperation, contributing greatly to K+ uptake. The sensitivity profileof this new system is in agreement with that of AtAKT1 (Bertl et al.,1997), suggesting that the pepper homolog CaAKT1 may greatlycontribute to K+ uptake under these conditions. On the other hand,the lack of inhibition of this new system by the presence of 10 mMNa+ and the strong inhibition by 1 mM Cs+ (Fig. 4) suggest that con-tribution of an AtCHX13 homolog may be low, because AtCHX13 ishighly sensitive to both Na+ and Cs+, and more sensitive to Na+ thanto Cs+ (Zhao et al., 2008).

Cs+ appears to be a potent inhibitor of high-affinity K+ uptakeboth in K+-starved plants (Fig. 1) and in K+-starved plants growingwith 50 �M K+ (Fig. 4), which may be explained by the sensitivity ofCaHAK1 (Rubio et al., 2008) and CaAKT1 (Bertl et al., 1997) to Cs+.CaHAK1 and CaAKT1 seem to be of great importance to maintaintissue K+ concentrations and plant growth at low K+, because whenthey are inhibited, root and shoot K+ concentrations (Fig. 3) andplant biomass (Fig. 2) were significantly reduced.

Here, we showed that the contribution of the systems for K+

uptake from diluted solutions was significantly affected by theregime of K+ supply employed to grow the plants. The contri-bution of CaHAK1 may be achieved by regulating the expressionlevel of the gene encoding it. It has been reported that CaHAK1expression takes place when the root K+ concentration is below thethreshold of 770 mmol K+ Kg−1

DW (Martínez-Cordero et al., 2005).

for 7 d in 50 �M K+ (Fig. 6), although the root K+ concentrationhas not increased significantly above that threshold (Fig. 3). Theobserved CaHAK1 repression may take place through changes inroot membrane potential, as described for the tomato homolog

1498 F. Rubio et al. / Journal of Plant Physiology 167 (2010) 1494–1499

Fig. 3. K+ concentrations in organs of plants grown in the presence of 50 �M K+

and inhibitors. Plants were grown as described in Fig. 2. After 7 d plant materialwas harvested and the K+ concentrations in roots and shoots determined. Data areaverages of 5 repetitions and error bars denote standard error.

Fig. 4. Effect of NH4+, Ba2+, Na+ or Cs+ on K+ absorption. Plants were grown as

described in Fig. 2. K+ absorption was calculated from the increase in total K+ contentin the plant after 7 d in the 50 �M K+ solution. The K+ absorption rate in the absenceotai

Lpitpto

FwocD

f inhibitors accounted to 0.68 �mol g−1DWroot min−1 and it was taken as 100% and

he absorption rates in the presence of inhibitors were referred to that control. Datare averages of 5 repetitions and error bars denote standard error. Different lettersndicate significant differences (p < 0.05) according to Tukey test.

eHAK5 (Nieves-Cordones et al., 2008). Hyperpolarized membraneotentials induce LeHAK5, and membrane depolarization represses

+

t. In K -starved pepper plants, a hyperpolarized membrane poten-ial may occur, which would induce CaHAK1. When 50 �M K+ isresent in the growth solution, membrane depolarization wouldake place, as would CaHAK1 repression. Rapid down-regulationf HAK genes by K+ re-supply has been also observed in other

ig. 5. Effect of NH4+, Ba2+ Na+ or Cs+ on K+ on K+ transport to the shoot. Plants

ere grown as described in Fig. 2. After 7 d in the 50 �M solution the percentagef K+ that was transported to the shoot with respect to the total K+ absorbed wasalculated. Data are averages of 5 repetitions and error bars denote standard error.ifferent letters indicate significant differences (p < 0.05) according to Tukey test.

Fig. 6. Expression of CaHAK1. The presence of CaHAK1 mRNA was determinedby RNA hybridization by using a probe synthesized from the full-length cDNA ofCaHAK1 in plants grown for 14 d in the absence of K+ or in the presence of 50 �M K+.Bottom panel shows etidium bromide staining of total RNA used for the analysis.

plant species (Armengaud et al., 2004; Nieves-Cordones et al., 2008;Shin and Schachtman, 2004), supporting the idea that high-affinityHAK transporters are only required when external K+ supply isextremely low (Nieves-Cordones et al., 2010; Qi et al., 2008). Theregulation of the contribution of CaAKT1 is more difficult to explainwith the available information. The genes encoding AKT1 chan-nels do not seem to respond to K+ supply (Lagarde et al., 1996;Martínez-Cordero et al., 2005). On the other hand, activation ofthe Arabidopisis AKT1 by the CBL1/9-CIPK23 complex seems to takeplace when plants are supplied with low K+ (Cheong et al., 2007;Lee et al., 2007; Li et al., 2006). A similar activation of CaAKT1by low K+ would explain its high contribution when plants aregrown at 50 �M K+, but it would not explain why CaAKT1 doesnot seem to contribute in K+-starved plants. It would be interest-ing to investigate whether, at extremely low external K+, whereCaHAK1 dominates, AKT1 is inactivated and how this inactivationis produced.

Interestingly, the percentage of K+ transported to the shoot inplants grown with 50 �M K+ was insensitive to NH4

+ but inhibitedby Ba2+ and Cs+ (Fig. 4). This indicates that an NH4

+-insensitive,Ba2+- and Cs+-sensitive system is mediating in this process. Ba2+

and Cs+ may enter the root stele and affect the systems involved in+

K release into the xylem for transport to the shoot, as for exam-

ple the shaker-like channel SKOR involved in K+ release into thexylem (Gaymard et al., 1998). Our results suggest that a pepperSKOR homolog may exist, and may be involved in K+ transport tothe shoot (Figs. 5 and 6).

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K

F. Rubio et al. / Journal of Plan

In conclusion, by using specific inhibitors, we provide datan the possible contribution of CaHAK1, CaAKT1 and a putativetCHX13 homolog to K+ uptake from diluted solutions in pepperlants, a species in which reverse genetics is not a straightforwardechnique. CaHAK1 seems to be major contributor to K+ uptakehen plants are grown with extremely low K+, and it mediates anH4

+- and Cs+-sensitive K+ uptake. At higher K+ concentrations,ithin the high-affinity range, CaAKT1 also contributes, consti-

uting Ba2+- and Cs+-sensitive K+ uptake. We also suggest theossibility that a pepper homolog to the Arabidopsis SKOR channel

s involved in K+ transport to the shoot. Further studies in modelpecies and crops will contribute to insights into the relevant sys-ems involved in K+ nutrition in plants.

cknowledgements

This word was funded by Grant 08696/PI/08 from Fundaciónéneca from Región de Murica, Spain and Grant AGL2009-08140rom Ministerio de Ciencia e Innovación of Spain to F.R. F.C. wasecipient of a JAE predoctoral fellowship from CSIC.

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