Characterization of a HKT-type transporter in rice as a

Characterization of a HKT-type transporter in rice as a general alkali cation transporter

Dortje Golldack1,2,Hua Su2,Francoise Quigley1,3,Uma R.Kamasani1,Carlos Mun?oz-Garay5,Enrique Balderas5,

Olga V.Popova2,John Bennett4,Hans J.Bohnert1,2,3,33,*and Omar Pantoja5

1Department of Biochemistry,2Department of Plant Sciences,and,3Department of Molecular and Cellular Biology,

The University of Arizona,Tucson,AZ85721,USA

4International Rice Research Institute,Division of Plant Breeding,PO Box933,Manila1099,Philippines

5Instituto de Biotecnologia,UNAM,Cuernavaca,Morelos62250,Mexico

Received26November2001;revised19March2002;accepted14May2002.

*For correspondence(fax217-333-5574;e-mail bohnerth@https://www.wendangku.net/doc/932987071.html,)

3Permanent address:Laboratoire de Gene?tique Mole?culaire des Plantes,UMR CNRS5575,Universite?Joseph Fourier,Grenoble,France

33Permanent address:Departments of Plant Biology and of Crop Sciences,University of Illinois,ERML196,1201W.Gregory Drive,Urbana,IL61801,USA 2Permanent address:Lehrstuhl fuèr Stoffwechselphysiologie und Biochemie der Pˉanzen,Universitaèt Bielefeld,Bielefeld,Germany

Summary

We report the characterization of rice OsHKT1(Oryza sativa ssp.indica)homologous to the wheat K+/

Na+-symporter HKT1.Expression of OsHKT1in the yeast strain CY162defective in K+-uptake restored

growth at mM and m M concentrations of K+and mediated hypersensitivity to Na+.When expressed in

Xenopus oocytes,rice OsHKT1showed uptake characteristics of a Na+-transporter but mediated

transport of other alkali cations as well.OsHKT1expression was analysed in salt-tolerant rice Pokkali

and salt-sensitive IR29in response to external cation concentrations.OsHKT1is expressed in roots and

leaves.Exposure to Na+,Rb+,Li+,and Cs+reduced OsHKT1transcript amounts in both varieties and,in

some cases,incompletely spliced transcripts were observed.By in situ hybridizations the expression of

OsHKT1was localized to the root epidermis and the vascular tissue inside the endodermis.In leaves,

OsHKT1showed strongest signals in cells surrounding the vasculature.The repression of OsHKT1in the

two rice varieties during salt stress was different in various cell types with main differences in the root

vascular tissue.The data suggest control over HKT expression as a factor that may distinguish salt

stress-sensitive and stress-tolerant lines.Differences in transcript expression in space and time in

different lines of the same species appear to be a component of ion homeostasis correlated with salt

sensitivity and tolerance.

Keywords:HKT1,Rice,Salinity tolerance.

Introduction

Physiological evidence for different K+uptake systems in higher plant roots has been presented long ago,based on the kinetics of K+-inˉux which showed both high-and low-af?nity components(Epstein et al.,1963).More recently, these K+uptake systems have become amenable to molecular analysis by the isolation and functional charac-terization of cDNAs for transmembrane proteins involved in plant K+homeostasis(Anderson et al.,1992;Cao et al., 1995;Fu and Luan,1998;Kim et al.,1998;Maathuis et al., 1997;Muèller-Roèber et al.,1995;Santa-Maria et al.,1997; Schachtman and Schroeder,1994;Schachtman et al.,1992;Sentenac et al.,1992;Su et al.,2001;Zimmermann et al., 1998).The existence of several types of K+-transporting membrane proteins has been documented,among them AKT/KAT-type channels,HKT-type transporters,and HAK/ AT/KUP-like transporters(Maser et al.,2001).

Based on their kinetic behaviour,the AKT/KAT family of inwardly rectifying K+channels seems to represent a low-af?nity component of the uptake machinery although this assignment has recently been questioned(Hirsch et al., 1998;Spalding et al.,1999).Expression of the root-speci?c K+channel AKT1was reported for both m M and m M

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external K+concentrations(Lagarde et al.,1996).Also, growth of an Arabidopsis AKT1-mutant was reduced at m M K+concentrations indicating that AKT-type K+channels are not only involved in low-but also in high-af?nity root K+ uptake(Spalding et al.,1999).The selectivity of these channels for K+over other alkali metal ions is variable,but most are characterized by ef?cient Na+exclusion and their contribution to sodium uptake seems to be minimal (Amtmann and Sanders,1999;Blumwald et al.,2000; Maathuis et al.,1997).

Another family of K+transporters is represented by the HAK/AT/KUP-type proteins with homology to bacterial KUP and HAK1from Schwanniomyces occidentalis (Banuelos et al.,1995).Homologues have been isolated from barley,ice plant and Arabidopsis thaliana(Fu and Luan,1998;Kim et al.,1998;Santa-Maria et al.,1997;Su, 2001).These transporters show low-as well as high-af?nity K+uptake characteristics when expressed in yeast (Quintero and Blatt,1997).

A member of a presumably high-af?nity K+-uptake system has been described in the wheat HKT1protein (Rubio et al.,1995;Schachtman and Schroeder,1994).It could also be a major route for the entry of Na+into roots, according to its speci?city as a K+/Na+symporter(Rubio et al.,1995).Because many conclusions about HKT1derive from functional analysis in yeast and Xenopus oocytes, HKT's action in cells of the root may be different,may be restricted to speci?c cells,or its activity may be masked or counteracted by the presence of other transport systems (Maathuis and Sanders,1995,1997;Maathuis et al.,1997; Tanner and Caspari,1996).The presence of wheat HKT1 leads to Na+uptake in high-Na+media and,if expressed and active in roots,could signi?cantly increase Na+ toxicity.Thus,a characterization of HKT-type transcripts and proteins in additional models is appropriate.

With the characterization of three K+-uptake systems, views about ion uptake are changing.Several protein subfamilies of the AKT and KUP families exist,each with different isoforms,and seemingly with gradual transitions in their kinetic and biochemical characteristics.In addition, ion speci?city varies not only between the members of a family in one species,but also for orthologous family members among different species.

Our interest in K+uptake systems is based on the goal of understanding the mechanisms that confer salinity toler-ance to land plants.We have concentrated on the rice homologue of wheat HKT1because of the large dataset available on rice salinity stress responses based on physiological experiments(Flowers and Yeo,1995; Garcia et al.,1995;Yeo et al.,1990).Also,a number of varieties and breeding lines are available that are charac-terized with respect to K+uptake and how salinity stress tolerance is acquired.We decided to utilize the Na+uptake characteristics of two rice cultivars,the salt-tolerant line Pokkali and the salt-sensitive line IR29,for a comparative analysis of the function of rice HKT1.We have character-ized a transcript,OsHKT1,encoding a plasma membrane protein with homology to the wheat high-af?nity K+ transporter HKT1.As we will show,Pokkali and IR29 exhibit signi?cant differences in their ability to take up or exclude Na+.Expression of OsHKT1in both lines is affected by the presence of high concentrations of alkali ions,which repress the HKT transcript.However,repres-sion was less pronounced in the root and leaf vascular tissues of the salt-sensitive IR29compared to amounts present in the salt tolerant Pokkali.A similar conclusion has recently been drawn by Horie et al.(2001)in a comparison between lines Nipponbare and Pokkali. Taking their analyses and our results,that include different characterizations,it seems that HKT1gene expression characteristics in the two varieties are signi?cantly correl-ated with ion homeostasis.

Results

Rice varieties with different K+-uptake characteristics

K+and Na+accumulation was analysed in two lines of indica rice(Oryza sativa):IR29(salt-sensitive)and Pokkali (salt-tolerant),adapted to either4m M K+,0.1m M K+,or medium without K+added(referred to as0m M K+in the following)that contained10±16m M K+from impurities in other salts(Figure1).Under control conditions,leaf Na+ and K+concentrations between the varieties were not signi?cantly different when the plants were grown at0,0.1, and4m M of K+,respectively.Pokkali and IR29showed, however,different K+and Na+uptake characteristics when the plants were salt-stressed at different external K+ concentrations.Both lines accumulated Na+very ef?-ciently in leaves but not roots when salt-stressed.When adapted to4m M and0.1m M external K+,IR29plants accumulated more than1000m mol g±1FW Na+in leaves following72h of salt treatment whereas Pokkali plants excluded Na+and had a leaf Na+contents of about30% compared to IR29.In the absence of K+in the medium Pokkali accumulated less Na+than IR29during short-term treatment of24h NaCl.After72h of salt treatment, however,the leaf Na+contents were similar in both rice lines with approximately700m mol g±1FW.Na+uptake was generally less in the salt tolerant rice line Pokkali,an effect that was dependent on the presence of external K+in the nutrition medium.

A rice HKT1-homologue

Based on the nucleotide sequence of a partial rice cDNA clone(Cornell Collection RZ405)homologous to the wheat high-af?nity K+transport protein HKT1(Accession No.

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U16709),we used RT-PCR ampli?cation,cDNA library screening and 5￠RACE extension to obtain a full-length transcript of the rice homologue.The cDNA sequence of OsHKT1(AF313388)isolated from the rice line IR36is 1827nucleotides in length.The 5￠UTR of OsHKT1is 60nucleotides in length and contains a consensus sequence for eukaryotic translation start sites (Kozak,1984).The

3￠-end is 173nucleotides in length and includes several putative polyadenylation sites.OsHKT1includes an open reading frame encoding a protein of 530amino acids for which a location in the plasma membrane is predicted (http://psort.ims.u-tokyo.ac.jp).The theoretical pI of OsHKT1is 9.43and the molecular weight is 59.3kDa (http://www.expasy.ch).An analysis of the reading frame predicted a protein characterized by 12hydrophobic domains,which ?ts the four-MPM model proposed by Durell et al .(1999).Kato et al .(2001)have convincingly shown this membrane topology for AtHKT1.A comparison with the wheat HKT1deduced protein sequence indicated co-linearity of the two proteins with high sequence identity in the hydrophobic regions.The deduced amino acid sequence of rice OsHKT1is 65%identical to that of wheat HKT1(Schachtman and Schroeder,1994),with an overall similarity of 77%.OsHKT1and the Arabidopsis homologue AtHKT1(Uozumi et al .,2000)show 39%sequence identity and 56%sequence homology.Recently,DNA sequences of OsHKT1have been obtained from japonica rice Nipponbare and indica line Pokkali (Horie et al .,2001).The deduced amino acid sequences of both are identical to OsHKT1isolated from line IR36reported here.Between Nipponbare and the two indica lines four nucleotides are different in the coding region and the 5￠non-coding region of Nipponbare shows a four-nucleotide deletion.All rice HKT sequenced so far contains two short introns.

Southern-type hybridizations (Figure 2)indicated three different DNA fragments with sequence homology to the cDNA of the isolated OsHKT1in the rice Pokkali and IR29genomes.Identical hybridization patterns were obtained from the genomes of both rice cultivars.Based on a genome size of 560Mb for rice,a copy number reconsti-tution also indicated a signal strength equivalent to at least two copies.Also,restriction digests with several enzymes,the sites of which are not present in the sequenced cDNA,highlighted gene-size fragments whose intensity indicated at least two HKT1-homologous sequences in the rice genome.In fact,two different cDNAs with 91%identity to each other have been isolated from line Pokkali (Horie et al .,2001).

Rice HKT1complements yeast mutants defective in K +uptake

Yeast complementation was used to monitor the potential function of OsHKT1in K +transport (Figure 3).A mutant,CY162,which is de?cient in TRK1and TRK2(Ko and Gaber,1991)grows at 100m M KCl.This strain,as well as a strain transformed with vector only,is unable to grow on 10m M KCl.When the OsHKT1coding region under control of the inducible Gal1-promoter was transformed into CY162,cells were unable to grow at low K +in the absence of galactose (Figure 3a).Induction of the promoter

by

Figure 1.K +and Na +uptake in different varieties of rice.

Plants were grown in hydroponic tanks for 3weeks and were then stressed by the addition of 150m M NaCl for 24and 72h.Plants were grown without (a),100m M (b),4m M K +(c).Filled bars,K +in roots;open bars,Na +in roots;striped bars,K +in leaves;stippled bars,Na +in leaves.The second oldest leaf was used in all cases with standard deviation (n =3).Media were controlled for the amount of ions by HPLC.The medium to which no K +was added contained according to HPLC analyses (Adams et al .,1992)between 10and 16m M K +from impurities in the other ions used.Inserts show amounts of potassium (?lled bars)and sodium (open bars)in the roots drawn to a different scale.

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galactose (arginine/phosphate-based medium;Rodriguez-Navarro and Ramos,1984)allowed growth in as low as 300m M KCl (Figure 3a).Addition of 150m M NaCl inhibited growth on plates and in liquid culture at 0.3m M KCl (Figure 3b).CY162transformed with the empty pYES2vector did not grow in the presence of galactose on agar plates and in liquid culture when KCl in concentrations of 7m M or less was present (not shown).

Electrophysiological characterization of OsHKT1

The electrophysiological characterization of OsHKT1was performed following methods established for wheat HKT (Gassmann et al .,1996;Rubio et al .,1995).Figure 4a shows that perfusion of the solutions containing 1.0m M Na +,0.3m M K +plus 1.0m M Na +or 0.3m M K +over an oocyte injected with water and held at the resting potential of ±40mV,did not cause any appreciable changes in the current levels.In contrast,perfusion of the same solutions

over an oocyte injected with OsHKT1and held at the resting potential of ±160mV induced the activation of inward currents.These currents were larger in the pres-ence of 1.0m M Na +plus 0.3m M K +than in the presence of 0.3m M K +or 1.0m M Na +alone.(Figure 4b).The expres-sion of OsHKT1caused polarization of the oocytes towards more negative potentials in the absence of Na +and K +.This is probably due to the release of Na +and/or K +

that

Figure 3.Rice OsHKT1complements a yeast TRK1/TRK2mutant.

The OsHKT1sequence cloned into a yeast expression vector (pYES2)was transferred into yeast strain CY162(Ko and Gaber,1991).Growth of CY162required 100m M KCl.

(a)Growth rate at different K +concentrations.(a)CY162,glucose-AAP medium,7m M K +(b)CY162::OsHKT1,glucose-AAP,7m M K +(c)CY162::OsHKT1,galactose-AAP,0.1m M K +(d)CY162::OsHKT1,galactose-AAP,1m M K +(e)CY162::OsHKT1,galactose-AAP,7m M K +.

(b)Effect of salt stress.(a)CY162::OsHKT1,glucose-AAP medium,0.3m M K +(b)CY162::OsHKT1,galactose-AAP,0.3m M K +,150m M NaCl;(c)CY162::OsHKT1,galactose-AAP,0.3m M K +

.

Figure 2.S outhern-type hybridizations of rice OsHKT1to rice genomic DNA.

Total rice DNA restricted as indicated was hybridized with a probe including the 3￠region of the coding region and 3￠UTR under high stringency conditions of washing.A difference in the Bam HI digestions of the IR29and Pokkali DNAs is due to incomplete digestion of the IR29DNA.A copy number reconstitution based on a rice genome size of 560Mb was used.The results indicated the presence of more than 2copies of OsHKT1-related sequences.

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has accumulated previously,as reported (Gassmann et al .,1996;Rubio et al .,1995),thus explaining the more negative holding potential for the injected oocyte (Figure 4b).We also characterized the HKT-induced currents by determin-ing the reversal potential under two conditions,maintain-ing extracellular K +constant at 1m M ,while varying Na +,and vice versa ,keeping external Na +at 1m M while changing K +(Rubio et al .,1995).With constant Na +(1m M ),increases in K +positively shifted the reversal potential by 21mV when K +was changed from 1m M to 10m M (Figure 5a).In contrast,when K +was kept constant at 1m M and Na +varied between 1m M and 10m M the reversal potential shifted positively by 15mV (Figure 5b).The wheat HKT1co-transporter is highly selective towards K +and Na +,allowing the movement of these two cations when present together,but it also mediates the movement of Na +in the absence of K +when Na +is found at millimolar levels (Gassmann et al .,1996;Rubio et al .,1995).The Arabidopsis homologue,AtHKT1was shown to function as a Na +uptake transporter independent of external K +when expressed in Xenopus oocytes (Uozumi et al .,2000).The AtHKT1-mediated current was not affected by the external K +concentration (Uozumi et al .,2000).Figure 5(c)shows the current-voltage relationships of a rice HKT1-injected oocyte exposed to alkali cations at concentrations

of 100m M .This ?gure demonstrates that in the presence of any of these cations,hyperpolarization of the oocyte induced inward currents in all cases,with a permeability sequence of Rb +>Cs +>Na +>K +>Li +.In contrast,the currents induced by the alkali cations in water injected oocytes were below 50nA with a permeability sequence Na +>K +>Li +>Rb +>Cs +(data not shown).These results are in contrast to reports on wheat HKT1(Gassmann et al .,1996;Rubio et al .,1995)where inward currents were recorded only in the presence of 100m M Na +(however,see Schachtman and Schroeder,1994).These results are also different from those reported by Horie et al .(2001)for japonica rice,cv.Nipponbare (Ni-OsHKT1)who reported a permeability sequence of Na +>Li +>K +b Rb +>Cs +.HKT1transcript abundance in different rice lines

Expression of OsHKT1in IR29and Pokkali depending on K +nutrition and salt stress was analysed by RT-PCR ampli?-cation with gene-speci?c oligonucleotide primers that were designed for the coding region and the non-coding region of the gene,respectively (Figure 6).In plants adapted to nutrition medium without K +,added expression of OsHKT1was detected in leaves and roots.Following treatment with 150m M NaCl for 24h,transcription of OsHKT1was reduced in leaves and roots of both rice lines.To study effects of increasing external K +on the expression of OsHKT1,the plants were adapted to medium containing 4m M K +.By RT-PCR,ampli?cation signals of OsHKT1were consistently detected in leaf (not shown)and root tissue of both rice lines (Figure 7).Two signals were obtained in transcripts from IR29roots.Sequencing of the additional (slightly larger)cDNA-fragment revealed this band as a differently spliced OsHKT1that included an intron of 291bp inserted between bp 1388and 1389of the cDNA (not shown;accession:AF450299,AF313388).The presence of this intron is con?rmed by the analysis of the rice genome sequence available of NCBI (HKT located on PAC clone #AP003726).The altered transcript includes two in-frame stop codons indicating that no functional OsHKT1protein will be translated.By exposure to 150m M NaCl for 24h OsHKT1signals were reduced in roots of both rice lines.

Cell-speci?c expression of rice HKT1

Cell-speci?city of expression of OsHKT1was studied by in situ hybridizations in the rice lines IR29and Pokkali adapted to 0m M K +.In root tips,IR29showed signals in the epidermis and exodermis,less in the cortex and strong expression in the vascular cylinder (Figure 8).In the vascular cylinder,signals were of approximately similar strength in endodermis,pericycle,phloem and xylem parenchyma cells.Pokkali showed little expression

of

Figure 4.Expression of rice HKT1in Xenopus oocytes induces inward currents in the presence of Na +and K +.

(a)Exposure of water-injected oocytes to 1mm Na +,1mm Na +plus 0.3mm K +or 0.3mm K +did not cause any current changes.

(b)Exposure of an oocyte injected with 25ng of rice HKT 1mRNA to solutions of 1mm Na +or 0.3mm K +caused the development of inward currents;presence of 1mm Na +plus 0.3mm K +caused an stimulation in the magnitude of the inward currents.

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OsHKT1in the vascular cylinder,but patchy expression in root epidermis,exodermis and adjacent cells of the cortex.In mature roots,expression in IR29was observed in epidermis,exodermis and the developing sclerenchymatic cells,as well as in the endodermis and the cells inside.Pokkali showed hybridization to the exodermis and weak signals in selected cells neighbouring the xylem vessels.For in situ hybridizations in leaves the region around the midrib of the second youngest leaf from 3-week-old plants was used.Signals were similar in IR29and Pokkali,with strongest expression in regions between the phloem and xylem and the transition from vasculature to mesophyll cells.

In root tips of both varieties,when adapted to medium without K +added,salt stress of 150m M NaCl decreased the signal to background levels within 24h (Figure 9).Salt stress did not abolish OsHKT1signals in the mature root as it did in the root tip.In IR29,signals in the epidermis and in part also in the exodermis disappeared or became weaker,but strong signals were still observed with scleren-chymatic cells,which still contain cytoplasm.In the vasculature,the endodermis stained weakly,but

signals

Figure 6.Quanti?cation of rice OsHKT1transcript amounts by RT-PCR in plants grown in nutrition medium without K +added.

Quanti?cation of OsHKT1by reverse transcription PCR.Left hand lanes are controls with RNA from seedlings,right hand lanes contain RNA from plants stressed for 24h in the presence of 150m M NaCl.TR,transcribed region of Hkt1;NTR,probe including the extreme 3￠-end of the coding region and part of the 3￠UTR;actin is a control ampli?cation with a rice actin gene

probe.

Figure 5.Expression of rice HKT1in Xenopus oocytes mediates alkali cation uptake.

(a)In the presence of 1m M Na +,the reversal potential of the HKT1-induced currents changed by 21mV per decade of extracellular K +concentration.

(b)While maintaining extracellular K +at 1m M ,a 10-fold variation in external Na +caused a change in the reversal potential of 15mV (C)I-V relationships from Os-HKT-injected oocytes exposed to 100m M solutions of different alkali cations.The results are representative traces.Similar results were obtained with >5oocytes from three different frogs.

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persisted in xylem parenchyma cells.OsHKT1expression in Pokkali was nearly exclusively restricted to the scleren-chymatic cells subtending the exodermis,and to xylem parenchyma cells in the centre of the vascular stele.In leaves,OsHKT1signals increased in the phloem of IR29plants under salt stress but not in Pokkali (not shown).Effects of alkali cations on the expression of OsHKT1Plants adapted to 0m M K +were treated with 150m M each of RbCl,LiCl,and CsCl,for 24h followed by RT-PCR ampli?cation of OsHKT1.The OsHKT1transcript level was reduced by treatment with all three cations in roots (Figure 10)and leaves (not shown)of Pokkali and by Li +-treatment alone in roots (Figure 10)and leaves (not shown)of IR29.Rb +and Cs +induced alternate splicing of OsHKT1in roots and leaves of IR29as it was similarly observed in roots of IR29plants adapted to 4m M K +(compare with Figure 7).Comparison of ion contents showed slightly increased K +,Li +and Rb +amounts in leaves of IR29treated with 150m M LiCl and 150m M RbCl,respectively,for 24h.In Pokkali,the K +content was not changed in comparison to control plants and the amounts of Li +and Rb +accumulated were lower than the concen-trations in IR29(Figure 11).Discussion Rice HKT1

We have isolated and characterized a homologue of wheat HKT1from the indica rice,IR36.OsHKT1expression in the salt-sensitive rice line IR29and the salt-tolerant line Pokkali was compared to begin to understand the possible role of this transporter in the homeostasis of monovalent alkali cations.OsHKT1shares 65%identity with the wheat homologue,39%identity with Arabidopsis AtHKT1,42%with Eucalyptus EcHKT1,and 46.5%with ice plant McHKT1(Su,2001).Recently,sequences of OsHKT1have been isolated from two rice varieties,the japonica rice,Nipponbare,and Pokkali (Horie et al.,2001).Both are identical to the IR36HKT1at the amino acid level,and only 4nucleotide differences exist in the coding region between

HKT1of the lines Nipponbare,Pokkali and IR29.OsHKT1appears to be encoded by a single gene in Nipponbare but a second transcript,OsHKT2,with 91%identity to OsHKT1,has been isolated from a Pokkali cDNA library (Horie et al.,2001).Our hybridizations indicated the existence of a small gene family of 2±3HKT-type sequences in both Pokkali and IR29.Possibly,HKT1copy number distinguishes indica and japonica rice.

Functional analyses of HKT-type transporters have been performed by heterologous expression in yeast and bac-terial strains de?cient in K +-uptake and in Xenopus oocytes.The wheat HKT1,for example,is highly selective for K +and Na +acting as a Na +-coupled K +uptake trans-porter.At high millimolar concentrations of Na +,low-af?nity Na +-uptake is mediated and high-af?nity K +-uptake is blocked (Gassmann et al .,1996;Rubio et al .,1995).For Arabidopsis AtHKT1expressed in Xenopus oocytes,large Na +currents were observed but no signi?cant uptake of K +Li +,Rb +,or Cs +occurred (Uozumi et al .,2000).Conversely,the Eucalyptus homologues EcHKT1and EcHKT2mediated alkali cation uptake with a higher permeability shown towards Na +and K +when heterologously expressed in Xenopus oocytes (Liu et al .,2001).Horie et al .(2001)reported OsHKT1identi?ed from rice Nipponbare and from indica Pokkali to be a Na +transporter and OsHKT2in Pokkali to be a Na +/K +-coupled transporter based on functional analyses in Xenopus oocytes and yeast.Interestingly,in the present study we found that the expression of OsHKT1conferred not only Na +hypersen-sitivity to a yeast mutant de?cient in the TRK1/TRK2K +transport systems but also K +uptake was restored enabling the mutant to grow on m M and m M K +con-centrations.In contrast to the experiments on yeast complementation with OsHKT1(Horie et al .,2001)we used a medium lacking NH 4+but containing arginine as the nitrogen source because competition of NH 4+with K +transport has been reported in yeast (Rodriguez-Navarro and Ramos,1984).These results indicate that the OsHKT1-mediated K +-transport in yeast is NH 4+-sensitive and may be inhibited by the ion.This is in contrast to the insensi-tivity to external NH 4+of wheat HKT1when the transporter is expressed in yeast (Santa-Maria et al .,

2000).

Figure 7.Expression of OsHKT1in rice adapted to 4m M K +.

OsHKT1transcription was monitored by reverse transcription PCR.Rc,root,control.Rs,roots from plants stressed with 150m M NaCl for 24h.

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Figure 8.In situ hybridization of OsHKT1cDNA to tissue sections.a,c,e,g,i,and k antisense;b,d,f,h,j,and l sense hybridizations.(a,b)IR29,root tip.(c,d)Pokkali,root tip.(e,f)IR29,mature root.(g,h)Pokkali,mature root.(i,j)IR29,leaf.(k,l)Pokkali,leaf.

The plants were adapted to nutrition solution without K +added.The bar in all prints represents 50m m.Abbreviations are:ep,epidermis;ct,cortex;vc,vascular cylinder;ph,phloem;xy,

xylem.

Figure 9.In situ hybridization of OsHKT1cDNA to tissue sections of salt-stressed rice.

a,c,e,and g antisense;b,d,f,and h sense hybridizations.(a,b)IR29,root tip,24h 150m M NaCl.(c,d)Pokkali,root tip,24h 150m M NaCl.(e,f)IR29,mature root,24h 150m M NaCl.(g,h)Pokkali,mature root,24h 150m M NaCl.

The rice plants were adapted to nutrition solution without K +added.The bars equal 50m

m.

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OsHKT1transcripts are expressed in speci?c cells and respond to alkali cations

OsHKT1was expressed in leaves and roots of IR29and Pokkali,but in situ hybridizations indicated differences in cell-speci?city.The expression of OsHKT1in the rice root in the two lines is observed in different cell layers.In the tip region,some signal was localized to the epidermis,but strongest expression in both varieties was seen in a layer of cells subtending the epidermis.Cortex cells had fewer signals in contrast to what has been reported for wheat HKT1(Schachtman and Schroeder,1994).A consistent difference between the rice varieties was seen for hybrid-ization to cells of the vascular tissue.In IR29,strong signals to all cells of the developing vascular cylinder,strongest to xylem parenchyma cells,contrasted with less OsHKT1-signal in the vasculature in Pokkali.In addition,Pokkali signals generally appeared in patches while IR29stained more uniformly.A possible interpretation of this cell speci?city would be general transport of K +towards the vasculature in the tip but lateral movement of K +in the mature root,consistent with our physiological data on ion uptake into the roots (see also,Schachtman and Schroeder,1994).In leaves,OsHKT1signals were found in cells adjacent to phloem vessels suggesting involve-ment of these transporters in K +-loading of the phloem.Based on kinetic studies in rye,White (1997)suggested

that,depending on the K +concentration in shoot and root phloem,the K +uptake rate into roots is regulated.

After NaCl stress,OsHKT1transcription was down-regulated in root-tips of both rice varieties as shown by in situ hybridizations.In mature roots the expression was reduced in the epidermis and vascular tissue with weaker transcript levels in Pokkali than in IR29.OsHKT RT-PCR fragments were then used to probe for expression differ-ences under salinity stress distinguishing the two lines.For the coding region of the gene,inhibition of the expression of OsHKT in the roots of both rice lines by salt treatment was shown.Signals from the untranslated gene-speci?c region of OsHKT1indicated that in IR29the speci?c expression of OsHKT1was not affected whereas in Pokkali the expression of the gene was inhibited.This comparison is valid because the 3￠untranslated regions of HKT are sequence-identical in IR29,Pokkali and Nipponbare in those segments that are found in all three genes (Horie et al .,2001).

OsHKT1transcripts were detected in leaves and roots of IR29and Pokkali after adaptation to both medium without K +and medium containing millimolar concentrations of K +.Horie et al .(2001)reported OsHKT1transcripts in the varieties Nipponbare and Pokkali to be higher in low potassium.In Arabidopsis ,transcription levels of AtHKT1were not affected by the addition of up to 100m M K +or 100m M Na +(Uozumi et al .,2000)whereas in roots

of

Figure 11.Accumulation of Li +and Rb +in rice.

The plants were grown in nutrition solution without K +added.They were either grown as non-stressed control plants or treated with 150m M LiCl (a)or with 150m M RbCl (b)for 24

h.

Figure 10.Effects of Rb +,Li +,and Cs +on the expression of OsHKT1.

Transcript amounts were quantitated by RT-PCR.Rice plants were adapted to nutrition solution without K +added and treated for 24h with 150m M Rb +,Li +,or Cs +.Rc,root,control.

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wheat and barley accumulation of HKT1mRNA was stimulated upon K+starvation(Wang et al.,1998).In barley this effect was most pronounced in young seedlings whereas at the age of8-to9-days HKT1expression was only weakly stimulated by K+starvation(Wang et al.,1998). It seems possible that reports on differences in the induction of HKT1by K+deprivation may have their basis in age-or development-speci?c regulation of these trans-porters(either amount,location in the membrane system or activity)and not in intrinsic qualities of the encoded proteins.

Effects of alkali cations on function and expression of OsHKT,respectively,were investigated by functional studies in Xenopus oocytes and by monitoring the transcript level of OsHKT1in rice plants treated with different cations.In Xenopus oocytes expressing OsHKT1 inward currents were induced not only by K+but all alkali cations tested with a permeability sequence Rb+>Cs+> Na+>K+>Li+,suggesting that OsHKT1functions as a general alkali cation transporter.Permeability of HKT1to all the alkali cations has been reported for wheat (Schachtman and Schroeder,1994)and Eucalyptus camal-dulensis(HKT1-2;Liu et al.,2001),although with a different permeability sequence.We have obtained similar results with the Mesembryanthemum crystallinum and Triticum aestivum HKT1homologues(unpublished results).The permeablity sequence we report here for rice HKT1con-trasts with that of Horie et al.(2001)who,using the same clone,showed a greater permeability to Na+compared with the other alkali cations.This difference is dif?cult to reconcile,however,our results were consistently observed in more than?ve oocytes from each of three different frogs with low SE(Figure5).Interestingly,Horie et al.(2001) proposed that the differences in selectivity between OsHKT1(a Na+transporter)and OsHKT2(a Na+/K+co-transporter)were due to a single amino acid change(Gly-88in OsHKT2,Ser-88in OsHKT1).However,this observa-tion is not supported by our results with rice HKT and those of Feirbain et al.(2000)and Liu et al.(2001)for the two HKT orthologues from E.camaldulensis,which func-tion as Na+/K+co-transporters where the equivalent amino acid corresponds to Ser-95(EcHKT1)and Ser-94(EcHKT2). It may be argued that the currents reported here are smaller than those reported by Rubio et al.(1995), Gassman et al.(1996)and,in part,Horie et al.(2001). However,as a control we have injected the mammalian brain K+channel K v1.1into Xenopus oocytes and have recorded currents larger than10m A at30mV(data not shown),similar to those reported by other researchers for the same channel(Rolf et al.,2000).Also,current levels similar to those reported by us for the rice HKT homologue were recorded by Schachtman and Schroeder(1994)for wheat,Horie et al.(2001)for rice,and those by Feirbain et al.(2000)and Liu et al.(2001)for the two HKT1orthologues from E.camaldulensis.In root and leaf tissue of both rice lines OsHKT1expression was inhibited by exposure of plants to Na+and Li+.In Pokkali,Rb+and Cs+ induced down-regulation of the transcripts.In IR29,in the presence of millimolar concentrations of K+(representing excess),and in the presence of Cs+and Rb+,we observed a different RT-PCR fragment based on the presence of an intron.This suggests incomplete splicing as a possible mechanism by which transcripts might be retained in the nucleus or by which translatable transcripts are reduced under stress conditions.OsHKT1clearly acts as a general alkali cation transporter not only when expressed in Xenopus oocytes but also after expression in planta. Salt-dependent regulation of OsHKT1expression in rice Molecular mechanisms governing Na+uptake into plants are not well understood,but experimental data suggest non-speci?c inˉux through K+uptake systems(Amtmann and Sanders,1999;Blumwald et al.,2000;Lagarde et al., 1996;Maathuis and Sanders,1997;Maathuis et al.,1997; Schroeder et al.,1994).Several protein families could account for inadvertent Na+uptake-low-af?nity channels, dual-or high-af?nity transporters and voltage-indepen-dent cation channels(VIC).Another possible uptake vehicle are non-selective cation channels(Schachtman and Liu,1999;White,1997)and proteins like the wheat low-af?nity cation transporter LCT1(Schachtman et al., 1997).Many low-af?nity channels(AKT/KAT-type)are highly selective for K+,but other monovalent cations are not completely excluded(Blumwald et al.,2000;Maathuis et al.,1997;Schachtman et al.,1992).The expression of the homologue of inward-rectifying K+channels OsAKT1was not modi?ed in the root epidermis and endodermis of IR29 under salt stress conditions but was inhibited in Pokkali (D Golldack,F Quigley,CB Michalowski,U Kamasani and HJ Bohnert;unpublished).Dual-af?nity K+-transporters (HAK/AT/KUP-type)have recently been shown to be affected by Na+,or they may act as low-af?nity Na+ transporters(Fu and Luan,1998;Quintero and Blatt,1997; Santa-Maria et al.,1997;Su,2001).The K+uptake protein represented by HKT1from wheat has been shown to function as a K+/Na+-co-transporter.Its action as a low-af?nity uptake protein in the presence of100m M Na+ (Rubio et al.,1995)could explain Na+accumulation.Our results with rice showed a difference between two rice lines with respect to the expression of OsHKT in the root vascular tissue whereas expression in the root tip was identical.The difference correlated with salt sensitivity in IR29and tolerance in Pokkali,indicating a possible involvement of OsHKT1in uptake and long-distance transport of cations in IR29that is probably realised in Pokkali to a much lower degree.As a response to alkali-salt stress OsHKT1transcription was down-regulated in roots

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and leaves of Pokkali,whereas that in IR29was either unchanged or down-regulated to a minor extent(Figures6 and8).If OsHKT1functions as a Na+transporter(Figures4 and5),our in situ and RT-PCR results(Figures6,8,9and10) suggest that regulation of OsHKT1by salt stress may be an important mechanism in salt tolerance.This view is supported by the consistent down-regulation of OsHKT1 caused by salt stress in the salt tolerant rice line Pokkali, leading to a lower level of Na+(Li+and Rb+)accumulation in planta,as compared to the amount taken up by the salt sensitive rice line IR29(Figures1and11).Further support comes from the recent detection of an Arabidopsis mutant in which salinity stress tolerance could be based on down-regulation(knockout)of the AtHKT1gene and protein(Rus et al.,2001).Our study suggests different cell-speci?city for HKT transcripts and,also,intraspecies speci?c differences under high salt conditions that distinguish the Na+-tolerant line Pokkali from Na+-sensitive IR29.Accordingly,HKT-type transporters may contribute to the differences in Na+-uptake behaviour of these rice lines in high salinity. Possibly of high signi?cance is that OsHKT seems to be involved in xylem loading and long distance transport of alkali cations from roots to shoots and in leaf phloem loading.The OsHKT transporter may thus determine how much Na+is ultimately taken up and transported to leaves in a feedback regulation inˉuencing other K+/Na+uptake systems in high salinity.The differences in OsHKT1 expression between IR29and Pokkali observed under salt stress conditions may be due to variations in promoter sequence,transcription factors,or in,as yet unknown, signalling events that control expression.A comparative analysis of such a control system in Pokkali and IR29will be necessary to gauge the relative importance of HKT1in K+homeostasis or Na+exclusion and uptake in rice.

Experimental procedures

Plant material

Seeds of rice(Oryza sativa L.indica)var.IR29and Pokkali were obtained from IRRI(Los Banos,Philippines),germinated in sand, transferred to aerated hydroponic tanks and grown for3weeks in all experiments(12-h light/dark at28°C/22°C)in controlled envir-onment chambers(Conviron,Winnipeg,Canada).Plants were grown in half-strength Hoagland's nutrient solution with K+ concentrations as indicated for the experiments.The addition of chelated iron was doubled.In experiments with reduced K+the NO3±counter ion was substituted by NH4+.For salt stress experiments,the nutrient solution contained150m M NaCl(or LiCl,RbCl or CsCl)for24h and72h,respectively.Unstressed control plants were grown in parallel and harvested at the same time.

Cloning and sequencing

A partial cDNA clone with homology to wheat HKT1was obtained from the Cornell Collection(clone RZ405)and sequenced with an automated ABI sequencer(model377,version 2.1.1). Ampli?cation of the5￠-end was done by5￠RACE-PCR(Gibco-BRL,Life Technologies,Inc.,Rockville,MD,USA).The full-length cDNA was assembled and cloned in the Xho I and Hin dIII restriction sites of pBluescript(Stratagene,La Jolla,CA,USA) and the entire cDNA-fragment was re-sequenced.The sequence of OsHKT1was deposited at GenBank(AF313388).

Southern hybridizations

Genomic DNA was extracted from IR29and Pokkali leaves according to Gustinich et al.(1991).DNA blotting and hybrid-ization were performed according to standard procedures (Sambrook et al.,1989).The probe,corresponding to nucleotides #283-1779of the full-length clone,was labelled with[32P]dCTP (Amersham-Pharmacia Biotech Inc.,Piscataway,NJ,USA)by random priming.After hybridization in2Q SSC,?lters were washed with0.2Q SSC at42°C for30min.For an estimation of the copy number in the rice genome(560Mb)plasmid DNA was loaded on the gels to produce signals equivalent to0.5±4copies.

In situ hybridizations

Root and leaf tissues were?xed with FAA,dehydrated and embedded.Root tips and segments approximately5cm from the tip were taken.Leaf sections were probed from the mid-section of the second youngest leaf of each plant.For tissue embedding Paraplast Plus(Fisher Scienti?c,Pittsburgh,PA,USA)was used. Sections10m m thick were mounted on poly-L-lysine coated slides.Sense and antisense RNA transcripts were synthesized by T3and T7RNA-polymerase from linearized pBluescript harbouring the cDNA(nucleotide#283-1779)using digoxigenin-UTP(Boehringer,Mannheim,Germany)as a label.Transcripts were hydrolyzed by alkaline treatment to an average length of200 nucleotides.In situ hybridizations were done according to Golldack and Dietz(2001)and signals detected by antidigoxigenin alkaline phosphatase-conjugated Fab fragments(Boehringer, Mannheim,Germany)and5-bromo-4-chloro-3-indolyl-phosphate and Nitroblue tetrazolium as a substrate.Microscopic analyses were carried out using an Axiophotˉuorescence microscope (Zeiss,Oberkochen Germany).Photographs were processed through Nikon Scan2.1and Adobe Photoshop.

RT-PCR

RNA from root and leaf tissue of rice(IR29and Pokkali)was isolated by guanidinium thioisocyanate extraction according to Chomczynski and Sacci(1987).cDNA was synthesized from total RNA using Superscript RT II(Gibco-BRL).Equal aliquots of cDNA were used as template for ampli?cation in50m l standard PCR reactions.A sequence speci?c forward primer(5￠-GCACTG-TCCAATGGAGAT-3￠;position1192-1209)and a reverse primer (5￠-TTCCTTCATCACTCCACC-3￠,position1493-1510)were used for the ampli?cation of a fragment of the3￠translated region of the sequence.A partial fragment of the3￠non-translated region was ampli?ed using the same forward primer and the reverse primer 5￠-TGCACTTGATGCACTTGC-3￠(position1626-1643).The PCR ampli?cations were carried out as described before(Golldack and Dietz,2001).A PCR-fragment with increased size from IR29 plants was cloned into PCR2.1TOPO(Invitrogen,Carlsbad,CA, USA)and sequenced.

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Analysis of ion contents

Root tissue and the second leaf of each plant were collected and ground in liquid nitrogen.After homogenization in ethanol: chloroform:water(12:5:3)and re-extraction with water,the aqueous phase was used for cation HPLC analysis(IonPac cation exchange;Dionex,Sunnyvale,CA,USA)(Adams et al.,1992). Alternatively,analyses of ion concentrations were performed with an inductively coupled plasma atomic emission spectrometer (ICP-AES,Perkin-Elmer,Boston,MA,USA)according to Brune et al.(1995).The results shown are means from5plants.

Yeast complementation

The full-length cDNA of OsHKT1was cloned in the Bam HI and Xho I restriction sites of pYES2(Invitrogen)under control of the inducible Gal1-promoter into S.cerevisiae CY162(Gietz et al., 1992;Ko and Garber,1991).Initial selections of transformants were done on glucose containing SC-agar plates without uracil supplemented with100m M K+.Positive transformants were replica-plated on galactose containing arginine/phosphate medium(Rodriguez-Navarro and Ramos,1984)without uracil containing7m M K+and selected a second time on arginine-phosphate top-agar plates without uracil supplemented with galactose and300m M K+.For growth studies,positive trans-formants were transferred to arginine/phosphate-based liquid medium of K+concentrations as indicated.Growth experiments, repeated at least three times with different transformants,were carried out in20ml aliquots and samples of0.3ml were taken to measure OD600(nm).The results shown are taken from one representative experiment.The plasmid was re-extracted from positive transformants and maintained in E.coli(d'Enfert et al., 1995).As a control,S.cerevisiae CY162was transformed with the pYES2vector without any insert.

Preparation of template DNA,in vitro transcription and capping of mRNA

The coding region of the OsHKT1gene was cloned into the pGEM-HE vector which contains a T7RNA polymerase promoter and5￠-and3￠-UTR of the Xenopus b-globin gene for enhanced expression(Liman et al.,1992).The plasmid DNA was puri?ed by CsCl gradient ultracentrifugation and linearized with Nhe I that cleaves the plasmid downstream of the inserted cDNA.OsHKT1 RNA was synthesized in vitro using the mCAP(TM)mRNA Capping Kit(Stratagene)a procedure,which tends to enhance the translation ef?ciency of RNA transcripts in Xenopus oocytes.

Two-electrode voltage clamping of Xenopus oocytes Oocytes injected with25ng of mRNA were used2±5days after injection for the recording of currents induced by the expression of HKT1with the two-electrode voltage clamp technique.Initially, the voltage electrode was introduced into the oocyte.After a stable membrane potential had been reached for several minutes, the current electrode was inserted.The electrodes were?lled with 1M KC1and the oocytes bathed with a solution containing6m M MgC12,1.8m M CaC12,10m M Tris/MES,pH5.5,with osmolality adjusted to240±260mOsmol kg±1with D-mannitol.K+and Na+ were added as glutamate salts in experiments where only these two cations were studied.In the experiments where the selectivity of HKT1was assayed,different alkali cations were added as chloride solutions.Recordings were obtained with a Dagan Oocyte Clamp ampli?er(Dagan,Minneapolis,MI,USA);data acquisition and analysis were done with program pCLAMP6 (Axon Instruments,Foster City,CA,USA).

Acknowledgements

We are grateful to Ms.W.Chmara(University of Arizona,AZ, USA)for HPLC analysis,to Ms.C.B.Michalowski(University of Arizona)for help with sequencing,to Ms.E.Reisberg(Universitaèt Wuèrzburg)for ICP-AES measurements.We also wish to thank Dr B.Barkla(UNAM)for help with the manuscript,and Dr A.Darszon (UNAM)for providing a two-voltage clamp ampli?er.We are indebted to Dr R.Gaber(University of Chicago)for providing yeast strains.The project has been supported in part by the Arizona Agricultural Experiment Station. D.G.acknowledges support by the Deutsche Forschungsgemeinschaft,Bonn, Germany.U.R.K.has been supported by a grant from the Rockefeller Foundation,New York,USA.CONACyT,Mexico,and the National Science Foundation(International Programs),USA, supported collaborative work(to O.P and H.J.B),CONACyT, Mexico,and the BMBF(International Bureau),Germany,sup-ported collaborative work(to O.P and D.G).O.P.received additional support from CONACyT(Grant33054-N).

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