Functional characterization of a potassium transporter gene NrHAK1 in Nicotiana rustica

Functional characterization of a potassium transporter gene NrHAK1 in Nicotiana rustica 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yangq@hit.edu.cn †E-mail:yangq@hit.edu.cn yangq@hit.edu.cn 2 2 Department of Life Sciences and Engineering, Harbin Institute of Technology, Harbin 150001, China Department of Life Sciences and Engineering, Harbin Institute of Technology, Harbin 150001, China Mudanjiang Tobacco Research Institute, Mudanjiang 157011, China Mudanjiang Tobacco Research Institute, Mudanjiang 157011, China The purpose of this study is to investigate the function of a novel potassium transporter gene ( NrHAK1 ) isolated from Nicotiana rustica roots using yeast complement and real-time PCR technique. The complementary DNA (cDNA) of NrHAK1 , 2 488 bp long, contains an open reading frame (ORF) of 2 334 bp encoding a protein of 777 amino acids (87.6 kDa) with 12 predicted transmembrane domains. The NrHAK1 protein shows a high sequence similarity to those of high-affinity potassium transporters in Mesembryanthemum , Phytolacca acinosa , Arabidopsis thaliana , and so on. We found that the NrHAK1 gene could complement the yeast-mutant defect in K + uptake. Among several tissues surveyed, the expression level of NrHAK1 was most abundant in the root tip and was up-regulated when exposed to potassium starvation. Moreover, the transcript accumulation was significantly reduced by adding 5 mmol/L NH 4 + to the solution. These results suggest that NrHAK1 plays an important role in potassium absorption in N . rustica . Functional characterization K + transporter Nicotiana rustica INTRODUCTION 1 Potassium (K + ) is an essential macronutrient in plants and functions as an important osmoticum and a cofactor for enzymes that affect protein synthesis, photosynthesis, and regulation of pH and the cation-anion balance. Root absorption of K + has been known as biphasic: a high-affinity system sustains K + accumulation in plants when external K + is in the micro-molar range; and a low-affinity transporter is responsible for K + uptake at the milli-molar K + concentrations (Epstein et al., 1963 1963 ). The high-affinity uptake mechanism has been shown to be inducible, whereas the low-affinity system may be constitutive (Glass and Dunlop, 1978 1978 ; Fernando et al., 1990 1990 ). There is a functional overlap between the high- and low-affinity uptake mechanisms (Hirsch et al., 1998 1998 ; Santa-María et al., 2000 2000 ). Absorption of K + may involve multiple transporter proteins, including AKT (arabidopsis K + transporter)-type channels, HKT (high-affinity K + transporter)-type transporters and HAK/KUP (high-affinity K + transporter/K + uptake permeases)-type transporters. HAK/KUP transporters were originally identified in Escherichia coli as KUPs (K + uptake permeases) (Schleyer and Bakker, 1993 1993 ). Later, their homologous genes were identified in soil-borne fungus Schwanniomyces occidentalis as HAKs (high-affinity K + ). Plant HAK/KUP transporters are homologous to the S . occidentalis HAK and the bacterium E . coli KUP (Santa-María et al., 1997 1997 ; Fu and Luan, 1998 1998 ; Kim et al., 1998 1998 ). HAK/KUP transporters are located in roots and shoots, and they may also be in the plasma membrane and the tonoplast (Rodríguez-Navarro, 2000 2000 ). Phylogenetic analyses indicate that plant HAK/KUP transporters can be classified into four clusters (Rubio et al., 2000 2000 ). Hydrophobicity plots show that the HAK/KUP protein contains 12 putative transmembrane domains (Kim et al., 1998 1998 ; Rubio et al., 2000 2000 ). The purpose of this study was to investigate the function of a novel potassium transporter gene ( NrHAK1 ) isolated from the root of Nicotiana rustica , which has higher potassium content than any other species of tobacco. We isolated and analyzed the NrHAK1 cDNA, performed functional complementation tests in yeast, and determined the NrHAK1 transcription levels under different ionic environments. MATERIALS AND METHODS 1 Strains and media 2 The E . coli strain DH5α was used for plasmidic DNA propagation. E . coli cells were cultured in Luria-Burtani (LB) medium with an appropriate antibiotic for selecting transformants. A Saccharomyces cerevisiae mutant strain trk1Δtrk2Δ , which was obtained from EGY48 ( MATα , his3 trp1 ura3-52 leu::pLeu2-LexAop6 ) (Invitrogen, CA, USA) by gene disruption, was the recipient strain for the yeast shuttle vector pPC87 plasmid. Yeast cells were grown in rich medium YPD [1% (w/w) yeast extract, 2% (w/w) peptone and 2% (w/w) glucose] or mineral medium APG (10 mmol/L arginine, 8 mmol/L phosphoric acid, 2% (w/w) glucose, 2 mmol/L MgSO 4 , 1 mmol/L KCl, 0.2 mmol/L CaCl 2 , trace minerals and vitamins, pH 4.0). The APG basic medium containing neither K + nor Na + was added as indicated, and was adjusted to pH 4.0 with diluted acetic acid. The effect of NH 4 + on the mutant strain was examined with ammonium as the nitrogen source in place of arginine. Solid media contained 1.5% (w/w) agar. Isolation of NrHAK1 2 Manipulation of nucleic acids was performed according to the standard protocols or the manufacturer’s instructions. The total RNA was isolated from the K + -starvated roots of Nicotiana rustica with the RNeasy Plant Mini Kit (Qiagen, Germany) and then treated with Dnase I (Promega, USA) to remove any remaining genomic DNA. The concentration of RNA was determined by the spectro-photometric analysis at the wavelength of 260 nm. As the template for polymerase chain reaction (PCR) amplification, the first-strand complementary DNA (cDNA) was synthesized from 2 μg total RNA with the SMART RACE cDNA Amplification Kit (Clontech, CA, USA). Degenerate primers for PCR were designed based on two conserved regions of HAK/KUP potassium transporters among several species. The primer for Kup1F was 5′-GCTGATGATAA(Y)GG(W)GA(R)GG(W)GG(W)AC(W)TTTGC-3′, deduced from the sense amino acid sequence GVVYGDL; and the primer for KuP3R was 5′-ATAAT(R)CTAAA(W)GT(W)GC(W)GAAAT-3′, corresponding to the sense amino acid sequence MFADLGHF. The gene-specific primers (GSPs) were designed based on degenerate PCR products. The 5′- and 3′-RACE PCRs were performed with the GSPs and the universal primers (UPs) supplied in the SMART RACE cDNA Amplification Kit (Clontech, CA, USA). A full-length cDNA of 2 488 bp was obtained and named NrHAK1 . The nucleotide sequence was deposited in the National Center for Biotechnology Information (NCBI) database (accession No. DQ535884). Amino acid sequence analysis 2 Multiple sequence alignments of amino acid sequences were generated by the CLUSTALW program ( http://www.ebi.ac.uk/clustalw/ http://www.ebi.ac.uk/clustalw/ ), and the phylogenetic tree was produced by Tree View based on complete amino acid sequences. The BOXSHADE program ( http://www.ch.embnet.org/software/BOX_form.html http://www.ch.embnet.org/software/BOX_form.html ) was used to analyze conserved amino acid regions after the initial alignment was obtained. Vector construction 2 The full-length NrHAK1 was amplified with the open reading frame (ORF) primers 5′-AAGAATTCATGTTACCCATGTTTGAAAA-3′ and 5′-AAAGAGCTCTTATACATGGTAAATCATTCC-3′ with the first-strand cDNA as a template. After the amplified gene was purified from agarose gel, the products were digested with Eco RI and Sac I and inserted into the yeast shuttle vector pPC87 plasmid that was digested in the same way. Fidelity of the constructs was checked by DNA sequencing, and the obtained plasmid was designated as pPCHAK. Expression of NrHAK1 in yeast 2 For the functional complementation analysis, the plasmid pPCHAK was introduced into the mutant yeast strain EGY4 trk1Δtrk2Δ with the lithium acetate method (Gietz et al., 1995 1995 ). The transformed yeast cells were selected in APG medium and then harvested and adjusted to OD 600 (optical density at 600 nm)=0.8 with double-distilled water. A serial of 10-fold dilutions of the strains starting at OD 600 =0.8 were prepared, and 5 μl of each dilution was spotted onto the solid APG basic medium supplemented with selected amounts of K + , Na + and NH 4 + provided as chloride salts. Cultures were grown at 30 °C for 7 d. Salt stress experiments 2 Tobacco seeds were surface-sterilized [5% (w/w) sodium hypochloride] and germinated in vermiculite soaked with modified one-third strength Hoagland solution containing 2.5 mmol/L KNO 3 , 1.0 mmol/L Ca(NO 3 ) 2 , 2 mmol/L MgSO 4 , 100 μmol/L EDTA-FeNa (ethylene diamine tetraacetic acid ferric sodium salt), 200 μmol/L KH 2 PO 4 , 50 μmol/L H 3 BO 3 , 0.5 μmol/L CuSO 4 , 2 μmol/L MnSO 4 , 0.5 μmol/L Na 2 MoO 4 , 0.1 μmol/L CaCl 2 , 2 μmol/L ZnSO 4 and 0.1 μmol/L KI. Experiments were carried out in a photoperiod with light:dark=16:8 (25 °C). Three-week-old seedlings were transferred to the hydroponic culture for ionic environmental experiments. Solutions of KNO 3 (2.5 mmol/L) and KH 2 PO 4 (200 μmol/L) were replaced by the ones with the same concentrations of Ca(NO 3 ) 2 and Ca(H 2 PO 4 ) 2 for K + deprivation treatment. In the salt stress experiment, 100 mmol/L Na + or 5 mmol/L NH 4 + was prepared by adding NaCl or NH 4 Cl to the modified one-third Hoagland solution up to the concentration indicated. Treatments were performed by transferring seedlings to each solution. Seedling roots were harvested after two days of treatments. Total RNA was isolated and subjected to the NrHAK1 expression analysis with real-time PCR. Rb + uptake experiments 2 The strains contained the empty vector and the vectors containing NrHAK1 cDNAs were grown in YPD medium. After 30-min K + starvation, the cells were suspended in a 2% (w/w) glucose, 10 mmol/L MES [2-( N -morpholine)-ethane sulphonic acid] buffer brought to pH 6.0 with Ca(OH) 2 . After the addition of RbCl to a final concentration of 0, 7.5, 15, 30, 60, 90, 120, or 180 μmol/L for 5 min, 1 ml sample cells in the absence or presence of two concentrations of K + (25 and 50 μmol/L) were taken, filtered through the 0.8-μm pore nitrocellulose membrane filters (Millipore, MA, USA), and washed with 20 mmol/L MgCl 2 . The filters were incubated overnight in 0.1 mol/L HCl, and the concentration of Rb + or K + was determined based on atomic emission of acid-extracted cells by a Perkin-Elmer 5 500 spectrophotometer (Boston, MA, USA). The average value of three independent experiments was reported. Real-time PCR 2 To generate the standard curves, the PCR products of NrHAK1 and tobacco β-actin (accession No. AB158612, used as a house-keeping gene for normalization) were purified with the QIA Quick Spin Purifying Kit (Qiagen, Germany). The concentration of purified DNA fragments was determined and further adjusted to 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 copies per microliter according to the OD 260 of PCR products and the amounts of four nuclei acids in the DNA fragment. The primers used in the qRT-PCR (real-time quantity PCR) were as follows: NrHAK1-F, 5′-CCTTATTGTGCCGTCATGCC-3′; NrHAK1-R, 5′-CTGAGATTGCAGGAGTAAGG-3′; ACT-F, 5′-GATCTTGCTGGTCGTGATCT-3′; and ACT-R, 5′-ACTTCCGGACATCTGAACCT-3′. The Ct (cycle threshold) values were estimated at a threshold value of 0.1, which was at least twice as high as the detection limit (corresponding to 10 times the standard deviation of the fluorescence in cycles 1~10). The NrHAK1 expression level in each sample was calculated as a ratio of the copy number of NrHAK1 to that of β-actin. Statistical analyses were performed in SPSS (version 10.0 for Microsoft Windows). Data were presented as the mean± SE (standard error) and P < 0.05 was considered statistically significant. After PCR, all reaction products were analyzed by agarose gel electrophoresis to confirm that only the expected PCR products were produced. RESULTS 1 Analysis of the NrHAK1 amino acid sequence 2 We first isolated a 0.73-kb cDNA fragment by RT-PCR, whose translated sequence showed a high sequence similarity to HAK/KUP potassium transporters of Mesembryanthemum , Phytolacca acinosa and Arabidopsis thaliana , and so on. The fragment was further extended by 5′- and 3′-RACE amplification, leading to a full-length cDNA of 2 488 bp containing a 2 334-bp ORF (777 amino acids). A BLASTx search for protein sequences related to NrHAK1 revealed a moderate sequence similarity to the HAK/KUP transporters. NrHAK1 appears to share a homology with HAK-type proteins in other plants: the similarity to Arabidopsis HAK/KUP transporters varies from 60% to 87% and the identity from 41% to 76%, respectively. NrHAK1 shows a particularly high homology to AtHAK8 (76% identity). The existence of closely related orthologs among different species suggests that HAK/KUP transporters are highly conserved not only in structure but also maybe in function. According to Rubio et al.( 2000 2000 ), all plant HAK/KUP transporters fall into four clusters. Fig. 1 1 shows the phylogenetic relationships among NrHAK1 and other HAK/KUP proteins in Arabidopsis thaliana , Phytolacca , Mesembryanthemum , and Hordeum vulgare , indicating that the NrHAK1 protein belongs to Group II. NrHAK1 appears to be in the same cluster of PaHAK1 , McHAK1 , AtHAK8 and AtHAK6 . The bootstrapped tree was produced by CLUSTALW with 1000 repetitions. A consensus tree for plant HAKs was generated with TREEVIEW software. The origins of the proteins are as follows: AtKup1 (At2g30070), AtKup2 (At2g40540), AtKup4 (At4g23640), AtKup3 (AAF19432), AtHAK5 (AAF19432), AtKup5 (At4g33530), AtHAK6 (At1g70300), AtKup7 (At5g09400), AtHAK8 (At5g14880), AtKup9 (At4g19960), AtKup10 (At1g31120), AtKup11 (At2g35060), AtKup12 (At1g60160), HvHAK1 (AF025 292), PaHAK1 (AAX13997), McHAK1 (AF367864), and NrHAK1 (DQ535884) Functional complementation of NrHAK1 in yeast mutants 2 The NrHAK1 cDNA was inserted into the yeast expression vector pPC87 and transformed into the mutant strain EGY4 trk1Δtrk2Δ . Our results show that all strains grew at similar rates in the medium containing 50 mmol/L KCl (Fig. 2a 2a ). The growth of mutant strains lacking the K + transporters trk1 and trk2 was rescued by the NrHAK1 expression in 0.1 mmol/L K + medium (Fig. 2b 2b ), whereas the mutants containing the empty vector pPC87 did not grow at the concentration of 0.1 mmol/L K + . Thus, we confirmed that NrHAK1 was able to support yeast growth at a concentration as low as 0.1 mmol/L K + in the culture medium, suggesting that the NrHAK1 protein functioned as a potassium transporter in N . rustica . Ten-fold dilution series of cell suspensions were prepared from yeast EGY48 [wild type (WT)], mutant yeast EGY4 trk1Δtrk2Δ transformed with NrHAK1 (pPCNrHAK) and mutant strain with empty pPC87 plasmid (pPC87). Suspensions were spotted on solid APG medium (pH 4.0) supplemented with (a) 50 mmol/L or (b) 0.1 mmol/L KCl. The plates were incubated at 30 °C for 7 d As shown in Fig. 3 3 , during potassium starvation treatments, the rate of Rb + influx in the K + -uptake-deficient yeast mutant with overexpressing NrHAK1 was fitted to a Michaelis-Menten function. We also found that the rate of Rb + influx in NrHAK1 transformant was four times larger than that in the control cells and had an apparent K m of 27.6 μmol/L for Rb + . Rb + uptake in the mutant yeast strain EGY48 trk1Δtrk2Δ transformed with NrHAK1 (NrHAK) or with the empty pPC87 plasmid (pPC87) Fig. 4 4 shows that the yeast-expressed NrHAK1 mediated the high-affinity Rb + uptake and it was competitively inhibited by K + at micromolar concentrations. Concentration dependence of the initial rates of Rb + uptake exhibited by the yeast potassium transport mutant transformed with the NrHAK1 cDNA in the absence or presence of two concentrations of K + (25 and 50 μmol/L) Growth of NrHAK1 transformant in the presence of Na + or NH 4 + 2 It has been reported that the non-specific uptake of potassium into cells is impaired when high extracellular concentrations of competing monovalent cations are available (Wang et al., 2000 2000 ). We investtigated the growth patterns of NrHAK1 transformant strains in the medium containing 100 mmol/L NaCl or 5 mmol/L NH 4 Cl. We found that NrHAK1 conferred on the yeast mutant a capacity to grow as fast as the wild-type strain in the NaCl medium (Fig. 5a 5a ), but not in the NH 4 Cl medium (Fig. 5b 5b ). This inhibition of the NrHAK1 -mediated K + uptake by ammonium is consistent with the ammonium inhibition in other plant roots (Rufty et al., 1982 1982 ). Ten-fold dilution series of cell suspensions were prepared from yeast EGY48 [wild type (WT)], mutant yeast EGY48 trk1Δtrk2Δ transformed with NrHAK1 (pPCNrHAK) and mutant strain with empty pPC87 plasmid (pPC87). Suspensions were spotted on solid APG medium (pH 4.0, containing 50 mmo/L KCl) supplemented with (a) 100 mmol/L NaCl or (b) 5 mmol/L NH 4 Cl. The plates were incubated at 30 °C for 7 d Specific expression of NrHAK1 in N . rustica 2 We performed the real-time PCR analysis on reverse-transcribed RNA isolated from roots (R), root tips (RT), young leaves (YL), old leaves (OL) and flowers (F) with the gene-specific primers. Through analyzing the expression data, we found that the NrHAK1 transcripts were most abundant in the root tip of seedlings (Fig. 6 6 ). Ten-μg aliquots of total RNA from roots (R), root tips (RT), young leaves (YL), old leaves (OL) and flowers (F) were subjected to real-time PCR quantification of NrHAK1 . The relative amounts were calculated and normalized with a ratio of copy number of NrHAK1 to that of β-actin. Data were presented as mean± SE of triplicate measurements ( n =15). The presence of the same letter on two columns indicates the absence of significant differences at P =0.05 Expression patterns of NrHAK1 under different ionic environments 2 Involvement of NrHAK1 in response to environmental conditions was tested by examining the effects of potassium, sodium, and ammonium on NrHAK1 expression in the root of N . rustica . Comparing the relative transcript abundance of treatments [(+K + +NH 4 + ) vs (+K + −NH 4 + ) and (−K + +NH 4 + ) vs (−K + −NH 4 + ), as shown in Fig. 7a 7a ], we found that the NrHAK1 transcription was sharply decreased by the addition of NH 4 + . This is consistent with previous studies suggesting that NH 4 + blocks the high-affinity K + uptake systems and has an inhibitory effect on plant growth, especially in low K + environments (Hirsch et al., 1998 1998 ; Santa-María et al., 2000 2000 ). Additionally, in plants grown in solution without NH 4 + (−K + −NH 4 + ), the NrHAK1 transcription level was reduced to 25% by adding 2.5 mmol/L K + (+K + −NH 4 + ). This indicates that transcription of NrHAK1 was more abundant in tobacco roots subjected to potassium starvation, which was supported by the comparison of (+K + +NH 4 + ) vs (−K + +NH 4 + ) and (+K + +Na + ) vs (−K + +Na + ) (as shown in Fig. 7b 7b ). Plants were treated with solutions lacking potassium in combination with added Na + or NH 4 + stresses, including absence (−K + ) or 2.5 mmol/L KCl (+K + ), absence (−Na + ) or 100 mmol/L NaCl (+Na + ) and absence (−NH 4 + ) or 5 mmol/L NH 4 Cl (+NH 4 + ), for 2 d. Total RNA extracted from roots was subjected to real-time PCR to analyze the expression of NrHAK1 . The relative transcripts were calculated and normalized with a ratio of the copy number of NrHAK1 to that of β-actin. Data are presented as mean± SE of triplicate measurements ( n =12). The presence of the same letter on two columns indicates the absence of significant differences at P =0.05 DISCUSSION 1 Plants have developed flexible strategies to deal with changes in their environment and to minimize adverse effects of nutrient deficiency and excess toxic ions. Adaptive response includes a significant alteration in gene expression, particularly for membrane transporters responsible for the uptake, efflux, and translocation of beneficial and toxic minerals (Maathuis et al., 2003 2003 ). Many inorganic nutrient transporters in plants are encoded by multi-gene families whose members often exhibit strongly overlapping expression patterns. Studies in plants and microorganisms have shown that these transporters have a wide variety of functional properties, including both high- and low-affinity K + transports (Fu and Luan, 1998 1998 ; Senn et al., 2001 2001 ). The difference in functional properties and spatial distributions of the transport systems is thought to allow the fine tuning of K + under varying environmental conditions in different plant tissues. Plant HAK/KUP transporter genes have been cloned and characterized from Arabidopsis thaliana ( AtHAK1-13 ), Hordeum vulgare ( HvHAK1-2 ), Oryza sativa ( OsHAK1-17 ), Lycopericon esculentum ( LeHAK5 ), Capsicum annuum ( CaHAK1-2 ) and Mesembryanthemum crystallinum ( McHAK1-4 ) (Su et al., 2002 2002 ). Most of the HAK transporters characterized so far are high-affinity K + ones, and only a few of them are low-affinity K + transporters (Santa-María et al., 1997 1997 ; Senn et al., 2001 2001 ). As recently proposed (Rubio et al., 2000 2000 ; Banuelos et al., 2002 2002 ), all HAK/KUP transporters can be grouped into four clusters. The cluster I genes such as OsHAK1 , HvHAK1 , CaHAK1 and AtHAK5 are involved in high-affinity K + uptake. For these transporters, the K m values for K + are 6, 27, 6.2 and 24 μmol/L, respectively (Banuelos et al., 2002 2002 ; Martínez-Cordero et al., 2004 2004 ; Gierth et al., 2005 2005 ), which are remarkably close to the K m range (4 to 35 μmol/L) for the high-affinity K + uptake systems (Epstein et al., 1963 1963 ). The affinity of cluster II HAK/KUP transporters for K + is less clearly defined. For this cluster, the barley transporter HvHAK2 has been reported to facilitate low-affinity transport with a K m of 5 mmol/L for K + (Rubio et al., 2000 2000 ; Senn et al., 2001 2001 ). Growth of the E . coli and yeast mutants is restored at micromolar K + concentrations by expressing cluster II genes such as AtKT/HAK/KUP1 , AtKT/HAK/KUP2 , AtKT/HAK/KUP4 and McHAK1 , McHAK2 , McHAK4 (Ahn et al., 2004 2004 ; Su et al., 2002 2002 ). Transgenic Arabidopsis suspension cells with over-expressing AtKUP1 showed an increased Rb + uptake at micromolar concentrations with an apparent K m of 22 μmol/L, indicating that AtKUP1 encodes a high-affinity K + uptake (Kim et al., 1998 1998 ). But when expressed in a yeast mutant, AtKUP1 was shown to mediate a dual-affinity K + transporter (Fu and Luan, 1998 1998 ). The expression of AtKUP3 , which belongs to cluster II, was up-regulated by K + starvation treatment (Kim et al., 1998 1998 ). Several studies have confirmed that different members of HAK/KUP transporters have tissue-specific expression patterns (Kim et al., 1998 1998 ; Rigas et al., 2001 2001 ; Langer et al., 2002 2002 ; Su et al., 2002 2002 ). Arabidopsis AtKT2 was found in roots and leaves (Quintero and Blatt, 1997 1997 ), whereas Arabidopsis AtKUP1 was predominantly expressed in roots. The barley HvHAK1 was only detected in roots. At the transcript level, many genes encoding K + channels and transporters are known to be regulated by external K + (Pilot et al., 2003 2003 ). The transcription of some members of the HAK/KUP family in Arabidopsis , Oryza sativa and Lycopersicon esculentum are up-regulated in response to K + deprivation (Kim et al., 1998 1998 ; Maathuis et al., 2003 2003 ; Qi et al., 2008 2008 ; Banuelos et al., 2002 2002 ; Wang et al., 2002 2002 ). High-affinity K + uptake activity of both Arabidopsis AtKUP1 and barley HvHAK1 is inhibited by millimolar concentrations of Na + when these transporters are expressed in yeast cells (Santa-María et al., 1997 1997 ; Fu and Luan, 1998 1998 ; Fulgenzi et al., 2008 2008 ). In Mesembryanthemum crystalline , HAK gene expression increases transiently in response to high salt concentrations (Su et al., 2002 2002 ; Rodríuez-Navarro and Rubio, 2006 2006 ). HAK K + transporters are NH 4 + sensitive for K + uptake in contrast to the K + channel that is insensitive to NH 4 + (Santa-María et al., 2000 2000 ). The inhibition of the CaHAK1 expression by external NH 4 + has been reported (Martínez-Cordero et al., 2005 2005 ). LeHAK5, a high-affinity K + uptake system in tomato plants, is induced in K + -starvated plants, and the presence of NH 4 + in the growth solution further increases LeHAK5 expression (Nieves-Cordones et al., 2007 2007 ). In this study, we found that the transcription of NrHAK1 in N . rustica was primarily accumulated in root tips and the expression of NrHAK1 was induced by K + starvation or high salt stress. The parsimony analysis of NrHAK1 with the Arabidopsis HAK/KUP family confirmed that NrHAK1 maintained a high amino acid sequence identity to AtHAK6 , a K + transporter belonging to cluster II. 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Nucleotide sequence and 3′-end deletion studies indicate that the K + -uptake protein kup from Escherichia coli is composed of a hydrophobic core linked to a large and partially essential hydrophilic C terminus . J Bacteriol , 1993 . 175 ( 21 ): 6925 - 6931 . 8226635 . Nucleotide sequence and 3′-end deletion studies indicate that the K+-uptake protein kup from Escherichia coli is composed of a hydrophobic core linked to a large and partially essential hydrophilic C terminus ME Senn , F Rubio , MA Banuelos , A Rodriguez-Navarro . Comparative functional features of plant potassium HvHAK1 and HvHAK2 transporters . Journal of Biological Chemistry , 2001 . 276 ( 48 ): 44563 - 44569 . 11562376 DOI: 10.1074/jbc.M108129200 http://doi.org/10.1074/jbc.M108129200 . Comparative functional features of plant potassium HvHAK1 and HvHAK2 transporters H Su , D Golldack , C Zhao , HJ Bohnert . The expression of HAK-type K + transporters is regulated in response to salinity stress in common ice plant . Plant Physiol , 2002 . 129 ( 4 ): 1482 - 1493 . 12177462 DOI: 10.1104/pp.001149 http://doi.org/10.1104/pp.001149 . The expression of HAK-type K+ transporters is regulated in response to salinity stress in common ice plant YH Wang , DF Garvin , LV Kochian . Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere-mediated signals . Plant Physiol , 2002 . 130 ( 3 ): 1361 - 1370 . 12428001 DOI: 10.1104/pp.008854 http://doi.org/10.1104/pp.008854 . Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere-mediated signals Z Wang , X Zhang , D Fedida . Regulation of transient Na + conductance by intra- and extracellular K + in the human delayed rectifier K + channel Kv1.5 . The Journal of Physiology , 2000 . 523 ( 3 ): 575 - 591 . 10718739 DOI: 10.1111/j.1469-7793.2000.00575.x http://doi.org/10.1111/j.1469-7793.2000.00575.x . Regulation of transient Na+ conductance by intra- and extracellular K+ in the human delayed rectifier K+ channel Kv1.5 We would thank Dr. Quan-hong YAO (Shanghai Academy of Agricultural Sciences) for kindly providing Saccharomyces cerevisiae strain EGY48 and the trk1Δtrk2Δ mutant strain. We would also thank Jing MA and Bao-xin YANG (Mudanjiang Tobacco Research Institute, Mudanjiang, China) for their technical assistance. 10.1631/jzus.B0820209 Project (No. 110200101008) supported by the State Tobacco Monopoly Administration, China 2008-07-007 2008-10-28 2008-10-11 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 09