expressions of oshkt1, oshkt2, and osvha are differentially regulated under nacl stress in...

Journal of Experimental Botany, of 12

doi:10.1093/jxb/erl199

RESEARCH PAPER

Expressions of OsHKT1, OsHKT2, and OsVHA aredifferentially regulated under NaCl stress in salt-sensitiveand salt-tolerant rice (Oryza sativa L.) cultivars

Md. Abdul Kader1,*, Thorsten Seidel1, Dortje Golldack2 and Sylvia Lindberg1

1 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080,SE 75007 Uppsala, Sweden2 Department of Biochemistry and Physiology of Plants, University of Bielefeld, D-33501 Bielefeld, Germany

Received 15 August 2006; Accepted 13 September 2006

Abstract

Under NaCl-dominated salt stress, the key to plant sur-

vival is maintaining a low cytosolic Na+ level or Na+/K+

ratio. The OsHKT1, OsHKT2, and OsVHA transporter

genes might play important roles in maintaining cyto-

solic Na+ homeostasis in rice (Oryza sativa L. indica cvs

Pokkali and BRRI Dhan29). Upon NaCl stress, the

OsHKT1 transcript was significantly down-regulated

in salt-tolerant cv. Pokkali, but not in salt-sensitive cv.

BRRI Dhan29. NaCl stress induced the expression of

OsHKT2 and OsVHA in both Pokkali and BRRI Dhan29.

In cv. Pokkali, OsHKT2 and OsVHA transcripts were

induced immediately after NaCl stress. However, in cv.

BRRI Dhan29, the induction of OsHKT2 was quite low

and of OsVHA was low and delayed, compared with that

in cv. Pokkali. OsHKT2 and OsVHA induction mostly

occurred in the phloem, in the transition from phloem

to mesophyll cells, and in the mesophyll cells of the

leaves. The vacuolar area in cv. Pokkali did not change

under either short- (5–10 min) or long-term (24 h) salt

stress, although it significantly increased 24 h after

the stress in cv. BRRI Dhan29. When expressional

constructs of VHA-c and VHA-a with YFP and CFP were

introduced into isolated protoplasts of cvs Pokkali

and BRRI Dhan29, the fluorescence resonance energy

transfer (FRET) efficiency between VHA-c and VHA-a

upon salt stress decreased slightly in cv. Pokkali, but

increased significantly in cv. BRRI Dhan29. The results

suggest that the salt-tolerant cv. Pokkali regulates the

expression of OsHKT1, OsHKT2, and OsVHA differently

from how the salt-sensitive cv. BRRI Dhan29 does.

Together, these proteins might confer salt tolerance in

Pokkali by maintaining a low cytosolic Na+ level and

a correct ratio of cytosolic Na+/K+.

Key words: Cytosolic Na+/K+ homeostasis, FRET efficiency,

NaCl stress, OsHKT1, OsHKT2, OsVHA, vacuole volumes.

Introduction

Salt stress is one of the most important abiotic stressesaffecting natural productivity and causes significant croploss worldwide. For plants, the sodium ion (Na+) is harmfulwhereas the potassium ion (K+) is an essential ion. Thecytosol of plant cells normally contains 100–200 mM of K+

and 1–10 mM of Na+ (Taiz and Zeiger, 2002); this Na+/K+

ratio is optimal for many metabolic functions in cells.Physico-chemically, Na+ and K+ are similar cations. There-fore, under the typical NaCl-dominated salt environment innature, accumulation of high Na+ in the cytosol, and thushigh Na+/K+ ratios, disrupts enzymatic functions thatare normally activated by K+ in cells (Bhandal and Malik,1988; Tester and Davenport, 2003; Munns et al., 2006).Therefore, it is very important for cells to maintain a lowconcentration of cytosolic Na+ or to maintain a low Na+/K+

ratio in the cytosol when under NaCl stress (Maathuis andAmtmann, 1999).The most important way to maintain a low cytosolic Na+

concentration is to minimize the influx of Na+ into thecytosol. Na+ influx can be restricted by means of selectiveion uptake. Non-selective cation channels (NSCCs) areproposed to be the dominant pathways of Na+ influx inmany plant species (Roberts and Tester, 1997; Tyermanet al., 1997; Buschmann et al., 2000; Davenport and Tester,

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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2000; Demidchik and Tester, 2002; Demidchik et al., 2002;Kader and Lindberg, 2005). However, the molecular iden-tity of these NSCCs is still unknown. It has also beensuggested that high-affinity potassium transporters (HKTs)mediate a substantial Na+ influx in some species, includingrice (Uozumi et al., 2000; Horie et al., 2001; Golldacket al., 2002a). In a recent study, it was demonstratedthat K+-selective channels play a significant role in Na+

uptake in a salt-sensitive rice, cv. BRRI Dhan29 (Kader andLindberg, 2005). With some exceptions, plant species havemultiple HKT transporters. In rice, eight functional HKThomologues (OsHKT1–4 and 6–9) have been identified(Garciadeblas et al., 2003). All of these functional genesencode proteins with distinct transport activities, whichmight be expressed in various tissues and/or organs. It hasbeen suggested that OsHKT1 is a Na+ transporter (Horieet al., 2001; Maser et al., 2002; Garciadeblas et al., 2003)and OsHKT2 a Na+/K+ co-transporter (Horie et al., 2001;Maser et al., 2002). OsHKT2 is believed to be the soleHKT gene in rice involved in K+ transport (http://www.ausbiotech.org/pdf/2006_Honours_booklet.pdf#search¼‘Identifying%20expression%20patterns%20of%20the%20HKT’; accessed 30 June 2006). OsHKT8 was very re-cently shown to be a Na+ transporter that contributes toincreased salt tolerance by maintaining K+ homeostasis inthe shoot under salt stress (Ren et al., 2005; Rus et al.,2005). This transporter is thought to be analogous to thefunction of the AtHKT1 gene in Arabidopsis, which isa Na+-transporter and, interestingly, plays a crucial role incontrolling cytosolic Na+ detoxification (Berthomieu et al.,2003; Rus et al., 2004; Sunarpi et al., 2005). Therefore, it islikely that the HKT gene family plays an important rolein Na+/K+ homeostasis in rice, even though some of itsmembers are evidently Na+ transporters.Another way for plant cells to deal with high cytosolic

Na+ is to transport it out of the cytosol, either into thevacuole or into the apoplast. Apoplastic sequestration ofNa+, however, plays no role in salt tolerance in rice, sincemost Na+ in the rice shoot arrives there through apoplasticstreaming (Yeo et al., 1999). Conversely, vacuolar com-partmentalization is an efficient strategy for plant cells tocope with salt stress (Fukuda et al., 1998, 2004; Apse et al.,1999; Blumwald, 2000; Chauhan et al., 2000; Hamadaet al., 2001; Tester and Davenport, 2003). When compart-mentalized into the vacuole, Na+ is no longer toxic to cells(Flowers and Lauchli, 1983; Subbarao et al., 2003), butis even advantageous for growth and osmotic adjustment(Zhu, 2003; Rodriguez-Navarro and Rubio, 2006), partic-ularly since the vacuole may occupy over 95% of thevolume of mature cells. In a recent study, it was alsodemonstrated that vacuolar compartmentalization is evi-dent under salt stress in the salt-tolerant rice, cv. Pokkali,whereas apoplastic sequestration of cytosolic Na+ isdominant in the salt-sensitive cv. BRRI Dhan29 (Kaderand Lindberg, 2005). The candidate protein for compart-

mentalizing Na+ in the vacuole is the tonoplast Na+/H+-antiporter, which is energized to do so by the vacuolarH+-ATPase (VATPase or VHA). Both VHA and pyro-phosphatase are generally required for the maintenanceof intracellular pH and ion homeostasis (Padmanabanet al., 2004). Through the hydrolysis of ATP, VHA generatesa proton motive force that energizes secondary transportacross the vacuolar membrane (like that of Na+) under highsalt conditions. VHA was shown to be important for salttolerance in Saccharomyces cerevisiae (Hamilton et al.,2002) and in many plant species as well (Golldack andDietz, 2001; Kluge et al., 2003a; Senthilkumar et al., 2005;Vera-Estrella et al., 2005).Like other salt-tolerant species, the salt-tolerant rice cv.

Pokkali contains less Na+ both in its roots and shoots undersalt stress than do salt-sensitive rice cultivars (Golldacket al., 2003; Kader and Lindberg, 2005). Several Na+/K+

-transporters could be involved in conferring the abilityto maintain a low cytosolic Na+ level in Pokkali. To clarifythe regulatory mechanisms involved in maintaining cyto-solic Na+ homeostasis in rice, the expression of OsHKT1,OsHKT2, and OsVHA were compared in both a salt-sensitive (cv. BRRI Dhan29) and a salt-tolerant (cv.Pokkali) rice at different time points under NaCl stress.The transcripts of these transporter genes were quantifiedusing real-time reverse transcription polymerase chainreaction (RT-PCR). Cell-specific expressions of the trans-porter genes were examined using in situ PCR to in-vestigate their specific roles in cytosolic Na+ accumulation/detoxification.

Materials and methods

Plant materials

Seeds of rice (Oryza sativa L. indica cvs Pokkali and BRRI Dhan29)were provided by the Bangladesh Rice Research Institute (BRRI,Gazipur, Bangladesh). The seeds were soaked in water for 48 h indarkness. Afterwards, they were germinated on vermiculite soaked inhalf-strength Hoagland’s solution. After a week, the seedlings weretransferred to hydroponic tanks containing half-strength Hoagland’ssolution. Seedlings were grown for another 2 weeks in a controlledenvironment chamber with day/night temperatures of 25/21 �C under14 h of light (300 lE m�2 s�1); humidity was approximately 50%.Afterwards, the plants were stressed by adding NaCl at a finalconcentration of 150 mM to the nutrient solution for between 1 h and72 h. Non-stressed control plants were grown concurrently andharvested at the same time.

RNA extraction and cDNA synthesis for qRT-PCR

RNA was isolated from the shoot and root tissues of rice cvs Pokkaliand BRRI Dhan29. For qRT-PCR, total RNA was isolated usingthe guanidinium isothiocyanate method. Synthesis of cDNA wasperformed as described by Golldack et al. (2002b). For PCR ampli-fication, the following sequence-specific forward and reverse oligo-nucleotide primers were used: 5#-GAGTCGTCTCAGAAATGA-3#and 5#-TGAACTTTCAGGCAGAAC-3# (OsHKT1), 5#-GAGTC-GTCTCAGAAATGA-3# and 5#-TTCTACGATTCAAAAGGC-3#

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(OsHKT2), and 5#-CTTCTGGCAATCTTGGAG-3# and 5#-CAGT-GTAGACGAAGTGCA-3# (OsVHA). The following conditionswere used for the PCR reactions: 1 cycle consisted of 1 min 30 s at94 �C, 1 min at 94 �C, 1 min at 55 �C, 2 min at 72 �C, and a finalextension of 10 min at 72 �C. OsHKT1 was amplified for 30 cycles,OsHKT2 for 45 cycles, and OsVHA for 28 cycles, for samples fromboth the roots and shoots of rice. The PCR products from RT-PCRamplifications were separated on 1.7% (w/v) agarose gels and stainedwith ethidium bromide. Photographic documentation was performedusing a gel documentation system (INTAS, Gottingen, Germany).

RNA extraction and cDNA synthesis for real-time RT-PCR

For real-time RT-PCR, total RNA was extracted using the RNeasyplant mini kit (Qiagen, Valencia, CA, USA) according to themanufacturer’s instructions. A total of 5 lg of RNA was reversetranscribed to cDNA with oligo (dT) 20 mer with a T7 promoter inthe 5# position and two randomized nucleotides in the 3# position,using SuperScript III reverse transcriptase (Invitrogen, Carlsbad,CA, USA). The cDNAwas diluted to 2 ng ll�1 and 5 ll of the dilutedcDNA (10 ng cDNA) was used as a template in each well forquantitative real-time PCR analysis. The cDNA was amplified usingSYBR Green PCR Master Mix (Finnzymes, Espoo, Finland) on theABI 7000 thermocycler (Applied Biosystems, Foster City, CA,USA). Primers for the actin gene were used as an internal control tonormalize the expression data for each gene. The following primersequences were used: 5#-ACACCCAATATTATTCCTCTTAA-3#and 5#-CGGGAATACGCTAAAGG-3# (OsHKT1), 5#-CAAAGG-CAGGTGAATCAAG-3# and 5#-CGATTCAAAAGGCCCTAA-3#(OsHKT2), and 5#-TTTCTTTTGCTACTGCCTTTATATTGC-3#and 5#-AGTGTAGACGAAGTGCATGAAGGA-3# (OsVHA).The PCR conditions were as follows: 50 �C for 2 min and 95 �C for

10 min, followed by 40 cycles of 95 �C for 15 s and 60 �C for 1 min.A dissociation kinetic analysis was performed at the end of theexperiment to check the specificity of annealing. Three replicationswere performed for each sample in each experiment; each experimentwas independently performed twice. Standard determination curveswere generated using serial dilutions of 50, 10, 2, and 0.4 ng cDNA ineach well for every experiment. Results were analysed according toMuller et al. (2002).

In situ PCR

Sections of leaves and roots were fixed with FAA, dehydrated, andembedded in Paraplast Plus (Fisher Scientific, Pittsburgh, PA, USA)as described by Golldack et al. (2002b). Microtome sections of 12 lmwere mounted on microscopic slides coated with aminoalkylsilane(S4651 silane-prep slides; Sigma-Aldrich, Germany). The tissuesections were deparaffinized and rehydrated, and then treated withproteinase K and RNase-free DNase I. Synthesis of cDNA wasperformed with oligo-dT primers and SuperScript II Reverse Tran-scriptase (Invitrogen, Breda, the Netherlands). PCR reactions wereperformed using the same gene-specific oligonucleotide primersused for the qRT-PCR reactions described earlier. The PCR con-ditions were as follows: 94 �C for 1 min 30 s in the first cyclefollowed by 1 min at 94 �C, 1 min at 55 �C, 2 min at 72 �C (with thesame number of cycles for each gene as in qRT-PCR), and a finalextension at 72 �C for 10 min. For signal detection Alexa Fluor 488–5-dUTP (Molecular Probes, Leiden, the Netherlands) was used asa label. Negative control reactions were performed without adding thegene-specific oligonucleotide primers to the PCR reactions. AfterPCR amplifications, the tissue sections were stained with 10 lMpropidium iodide for 10 min. Microscopic images were taken witha cooled CCD-Camera coupled to an Axioskop fluorescencemicroscope (Zeiss, Germany). In the microscopic images the specificsignals were indicated by green to yellow fluorescence and thenegative background signals by red.

FRET analysis

Protoplast isolation: Protoplasts from the leaves of both Pokkali andBRRI Dhan29 were prepared as described by Shishova and Lindberg(1999), but with some modifications. Leaves from approximately 10-d-old seedlings were sliced into 0.5 mm pieces and treated with 1%(w/v) cellulase (lyophilized powder; 10.0 units mg�1 solid) fromTrichoderma resei (Sigma, EC 3.2.1.4; Sigma-Aldrich, St Louis,MO, USA) and 0.6% (w/v) macerase (lyophilized powder; 0.6 unitsmg�1 solid) Maceroenzyme R-10 (EC 3.2.1.4; Serva Electrophoresis,Heidelberg, Germany) for 2 h. The protoplasts were washed twice inthe loading medium containing 0.5 M sorbitol (Sigma-Aldrich, StLouis, MO, USA), 0.1 mM CaCl2, 0.2% (w/v) polyvinylpolypyrro-lidone (PVP; Sigma-Aldrich, St Louis, MO, USA), and a buffer (pH5.5) containing 5 mM TRIS (Labassco, Germany) and 5 mM MES(Sigma-Aldrich, St Louis, MO, USA).

Protoplast transfection: Constructs of the V-ATPase subunits VHA-a, VHA-c, VHA-E, and VHA-B ofMesembryanthemum crystallinumfused to fluorescent proteins (Seidel et al., 2004, 2005) were used forthe transient transfection of rice protoplasts. The transfection wasperformed as previously described in the case of Arabidopsisprotoplasts (Seidel et al., 2004).

FRET measurement: Fluorescence resonance energy transfer(FRET) was measured using a confocal microscop (Leica SP2; LeicaMicrosystems, Heidelberg, Germany). Cyan fluorescence protein(CFP) was excited by the 458 nm line of an argon-ion-laser anddetected in the range of 470–510 nm as a control for the CFP crosstalkinto the FRET channel (530–600 nm detection and 458 nm excitationbandwidth). Yellow fluorescence protein (YFP) was detected in therange of 530–600 nm using the 514 nm line of the argon-ion-laser forexcitation. The YFP -signal was required for estimating YFP by directexcitation by the 458 nm laser line (Seidel et al., 2005).FRET-efficiency was calculated by relating the corrected emission

intensity in the FRET channel to the sum of the CFP emission and theFRET emission (Seidel et al., 2005):

E ¼ ðEmFRET � ð0:613EmCFPÞ=ðEmFRET þ EmCFPÞ

For each FRET-pair, the FRET-effect of 40 protoplasts wasmeasured in two independent experiments.

Measurement of vacuolar area

Protoplasts from shoots of cvs Pokkali and BRRI Dhan29 wereisolated as described above. Protoplasts were stained with 3 ll of5 mM 6-carboxyfluorescein diacetate (6-CFDA; Sigma-Aldrich, StLouis, MO, USA) in DMSO through PEG-mediated osmotic shock.Non-specific esterase activity in the vacuolar lumen cleaves thenon-fluorescent 6-CFDA to the fluorescent, pH-dependent dye, 6-carboxyfluorescein (6-CF; Preston et al., 1989). The 6-CF fluores-cence in the vacuoles was imaged using a laser scanning confocalmicroscop (Leica SP2; Leica Microsystems, Heidelberg, Germany).The dye was excited sequentially by the 458 nm and 488 nm linesof an argon-ion-laser. Images of the vacuoles were taken, show-ing a signal in the 500–530 nm range after passing through aLeica RSP500 short -pass filter (Leica). The areas of the vacuolarimages were measured using the Leica Confocal Software LCS Lite(ftp://ftp.llt.de/pub/softlib/LCSLite/).

Results

The transcript levels of OsHKT1 (a Na+-transporter),OsHKT2 (a K+-Na+ co-transporter), and OsVHA (an ener-gizer for tonoplast Na+/H+ antiporter under salt stress) were

Expressions of OsHKT1, OsHKT2, and OsVHA under salt stress 3 of 12


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quantified using real-time RT-PCR in the salt-sensitive ricecv. BRRI Dhan29 and the salt-tolerant rice cv. Pokkali after0, 1, 6, 24, 48, and 72 h of salt stress with 150 mM NaCl.The cell-specificity of these transcripts was also studiedusing in situ PCR. The conformational change and energytransfer efficiency of OsVHA upon NaCl stress was studiedby measuring FRET efficiencies between the peripheralstalk subunit VHA-E and the catalytic head subunit VHA-Band between the proteolipid VHA-c and the C-terminaltransmembranedomain ofVHA-a, respectively. The changesin vacuolar area in leaf cells (protoplasts) upon salt stresswere also investigated.

Expression of OsHKT1

OsHKT1 transcripts displayed variable expression withtime up to 72 h after 150 mM NaCl stress, in both root andshoot tissues of rice cvs Pokkali and BRRI Dhan29 (Fig. 1).There was clear up-regulation of the transcript in BRRIDhan29 in the root, 1 h after the stress, and in the shoot6 h after the stress. In the root tissue, the up-regulationincreased substantially 6 h after the stress. However, 6 hafter the stress, when the transcript displayed the highestinduction in both the root and shoot tissues, induction wasapproximately four times higher in the root and approxi-mately two times higher in the shoot than their respective

control levels. In both the root and shoot tissues, the up-regulation of the transcript compared with the controlexpression continued up to 72 h after the stress, but withsome variation in terms of degree of up-regulation. On theother hand, there was significant up-regulation of the trans-cript in cv. Pokkali only 24 h after the stress in both theshoot and root tissues, and the expression was less than 1-fold higher in both cases than the respective control levels.Although the transcript displayed an up-regulation com-pared with the control expression up to 72 h after the stressin the salt-sensitive cv. BRRI Dhan29, there was significantdown-regulation of this transcript in the salt-tolerant cv.Pokkali starting 48 h after the stress in both the shoot androot tissues. Seventy-two hours after the stress, the down-regulation of this transcript was approximately 62% in thePokkali shoot and approximately 72% in the Pokkali rootcompared with the control expression. In situ expressionof the OsHKT1 transcript confirmed its expression in boththe root and shoot tissues of BRRI Dhan29 and Pokkali(Figs 4, 5). Under NaCl stress conditions, in root tissue ofBRRI Dhan29 (Fig. 4), a strong signal was detected fromthe epidermis and vascular cylinder, but only a weak signalfrom the cortex. In root tissue of Pokkali, the signal wasweaker upon stress than the control signal. Under controlconditions, a strong signal in BRRI Dhan29 leaf tissuecame from the mesophyll cells and the phloem, a weakersignal from the epidermal cells, and no signal from thexylem; in Pokkali leaves, a strong signal came from themesophyll cells, phloem and xylem. After 48 h of NaClstress, the OsHKT1 transcript in BRRI Dhan29 disappearedfrom the epidermis and also, notably, from the phloem,while a somewhat higher signal came from the mesophyllcells. In Pokkali, however, down-regulation of the trans-cript was noted in all types of cells, most notably from thephloem, xylem, and mesophyll cells.

Expression of OsHKT2

Real-time analysis indicated a substantial increase in theOsHKT2 transcript immediately after stress with 150 mMNaCl in Pokkali, but not in BRRI Dhan29 (Fig. 2). Therewas an approximately 15-fold higher expression of OsHKT2in Pokkali shoottissue 1 h after the stress than in thecontrol. Although the up-regulation of OsHKT2 in Pokkalileaf tissue gradually decreased by the ensuing time points,at 72 h after stress it still represented a 158% higher ex-pression than that of the control. In the shoot tissue of BRRIDhan29, a significant up-regulation of the transcript wasfound at 6 h after stress, representing nearly a 2-fold higherexpression than that of the control. The up-regulationdecreased slightly afterwards up to 72 h after the stress.There was also an up-regulation of OsHKT2 in root tissueof Pokkali, which continued up to 72 h after the stress.However, the up-regulation in Pokkali roots was not ashigh as in the shoot tissue; it was less than 50% higher than

Fig. 1. Expression analysis of OsHKT1 by means of real-time RT-PCRamplification of RNA from root and shoot tissues of rice cvs Pokkali andBRRI Dhan29. The number of h (1, 6, 24, 48, and 72 h) elapsed aftergrowing plants under salt stress (150 mM NaCl) conditions is indicated.Amplifications were performed with specific primers for OsHKT1 andcompared with the expression under control conditions (no stress).



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the control level up to 48 h after the stress, and approxi-mately 177% higher 72 h after the stress. On the other hand,OsHKT2 was down-regulated in the root tissue of BRRIDhan29 up to 24 h after the stress (to nearly 50% of thecontrol expression), after which a slight up-regulation wasevident 48 h and 72 h after the stress.As demonstrated by in situ analysis (Fig. 4), under

control conditions OsHKT2 was mainly expressed in theepidermis and vascular cylinder of the root with some lowexpression in the cortex. After stress with 150 mM NaCl,a weaker signal was detected from all these types of rootcells from BRRI Dhan29, whereas a stronger signal wasobtained from Pokkali, in line with the real-time quantifi-cation of this transcript. Under control conditions, the ex-pression of OsHKT2 in leaf tissue was confined mainly tothe mesophyll cells (Fig. 5). After NaCl stress for 24 h,however, a very strong signal appeared from the phloemand the connecting area between the phloem and mesophyllcells. The signal in mesophyll cells was also somewhatstronger in stressed cells than in control cells.

Expression of OsVHA

As shown in Fig. 3, there was an induction of OsVHA inboth the roots and shoots of Pokkali 1 h after the stress,

which increased further 6 h after the stress. The inductionwas also noted in both the root and shoot tissues ofBRRI Dhan29, but not until 6 h after the stress, and toa lesser extent than in Pokkali. Induction of the transcript6 h after the stress was approximately four times higher inPokkali shoot tissue and nearly twice as high in BRRIDhan29 shoot tissue than their respective control expres-sions. Afterwards, the induction in both the cultivars startedto decrease with time. In the shoot tissue of both cul-tivars, the lowest induction occurred 48 h after the stress,which then gave some induction again at 72 h after thestress. On the other hand, in comparison with the shoottissue, the initial induction of OsVHA in root tissue waslower in both Pokkali and BRRI Dhan29, though 72 hafter the stress the induction was close to that of the shoot.Under control conditions, OsVHA in root tissue pro-

duced a strong signal from the epidermis and vascularcylinder, but a very weak signal from the cortex region inboth cultivars (Fig. 4). Moreover, after 24 h of stress with150 mM NaCl, the signal from the root vascular cylinderin BRRI Dhan29 became weaker, whereas the signal fromthe root epidermis in Pokkali was stronger. In leaf tissueof BRRI Dhan29, OsVHA was expressed under controlconditions in phloem cells, at the transition from phloemto mesophyll cells, and in the mesophyll cells (Fig. 5). On

Fig. 2. Expression analysis of OsHKT2 by means of real-time RT-PCRamplification of RNA from root and shoot tissues of rice cvs Pokkali andBRRI Dhan29. The number of h (1, 6, 24, 48, and 72 h) elapsed aftergrowing plants under salt stress (150 mM NaCl) conditions is indicated.Amplifications were performed with specific primers for OsHKT2 andcompared with the expression under control conditions (no stress).

Fig. 3. Expression analysis of OsVHA by means of real-time RT-PCRamplification of RNA from root and shoot tissues of rice cvs Pokkali andBRRI Dhan29. The number of hours (1, 6, 24, 48, and 72 h) elapsed aftergrowing plants under salt stress (150 mM NaCl) conditions is indicated.Amplifications were performed with specific primers for OsVHA andcompared with the expression under control conditions (no stress).



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the other hand, in leaf tissue of Pokkali, no signal wasdetected from the phloem cells or from the transition fromthe phloem to mesophyll cells; the expression was insteadconfined to the mesophyll cells alone. After 1 h of stresswith 150 mM NaCl, however, a very strong signal wasdetected in the Pokkali leaf tissue from the phloem cellsand from the transition from the phloem to mesophyllcells, along with comparatively stronger expression in themesophyll cells. A stronger signal from the aforesaid cellswas also found in BRRI Dhan29 leaf tissue, but not to thesame extent as in the Pokkali leaf tissue.

Effect of salt stress on vacuolar area

Vacuoles, comprising up to 95% of cell volume in maturecells (Epimashko et al., 2004), are thought to be a good sinkfor the compartmentalization of cytosolic Na+. So changesin vacuole area could be evident under salt stress. However,no significant change were found in the vacuolar area in thesalt-tolerant cv. Pokkali upon salt stress, after either short-term (5–10 min) or long-term (24 h) treatment (Fig. 6).On the other hand, compared to the control (91.1616.06),the vacuolar area in the salt-sensitive cv. BRRI Dhan29

decreased (62.62610.5) after 5–10 min of NaCl stress, butthen increased (115.1660.81) after 24 h of stress. Thevacuolar area in unstressed tissue was the same in bothcultivars: 102.466.26 lm2 in Pokkali and 91.1616.06 lm2

in BRRI Dhan29.

Effect of salt stress on VHA structure

VHA is a protein complex of 13 different subunits witha bipartite structure similar to that of F-ATP -synthases(Seidel et al., 2005). The membrane integral sector, V0, iscomposed of subunits VHA-a, c, d, and e and the cyto-plasmically exposed V1 sector contains the subunits VHA-A to VHA-H. Sector V0 catalyses the proton transport,whereas sector V1 is involved in ATP binding and hy-drolysis. To test whether salt has an effect on the statorstructure of the VHA, the FRET-efficiency between theperipheral stalk subunit VHA-E and the catalytic headsubunit VHA-B was measured. No changes were identifiedas due to NaCl stress in either cv. Pokkali (48.3461.73and 46.7661.32) or cv. BRRI Dhan29 (38.5761.86 and40.8162.26). The VHA of cv. Pokkali displayed a signif-icantly (t test; P¼0.000128) higher FRET efficiency

Fig. 4. Cell-specific expression analysis through in situ PCR of OsHKT1 in rice cv. BRRI Dhan29 root (a: control; b: stressed with 150 mM NaCl for24 h) and Pokkali root (c: control; d: stressed with 150 mM NaCl for 24 h), OsHKT2 in rice cv. BRRI Dhan29 root (e: control; f: stressed with 150 mMNaCl for 48 h) and Pokkali root (g: control; h: stressed with 150 mMNaCl for 48 h), and OsVHA in rice cv. BRRI Dhan29 root (i: control; j: stressed with150 mM NaCl for 1 h) and Pokkali root (k: control; l: stressed with 150 mM NaCl for 48 h). Abbreviations: ep, epidermis; ct, cortex; vc, vascularcylinder. The bar in all plots represents 50 lm. The specific signal is shown by green to yellow fluorescence of Alexa Fluor 488–5-dUTP (MolecularProbes, Leiden, the Netherlands), as indicated by the white arrow.



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between VHA-E and VHA-B than BRRI Dhan29, in-dicating structural differences between these two enzymes.To investigate the structural flexibility of the transmem-brane sector, V0, of the V-ATPase, the FRET efficiencybetween the proteolipid VHA-c and the C-terminal trans-membrane domain of VHA-a was measured. As shown inFig. 7, under control conditions the FRET efficiency wassimilar in Pokkali (46.1661.84) and BRRI Dhan29(44.661.82); however, the efficiency of energy transferincreased significantly (t test, P¼0.00154) due to NaCl

stress in cv. BRRI Dhan29 (51.5461.45) and decreaseda little in cv. Pokkali (43.4361.76).

Discussion

The present study detected the induction of OsHKT1in both the root and shoot tissue of BRRI Dhan29 underhigh NaCl conditions, although the degree of inductioncompared with the control expression varied with time.

Fig. 5. Cell-specific expression analysis through in situ PCR of OsHKT1 in rice cv. BRRI Dhan29 leaf (a, c: control; b, d: stressed with 150 mM NaClfor 24 h) and Pokkali leaf (e: control; f: stressed with 150 mMNaCl for 24 h), OsHKT2 in rice cv. BRRI Dhan29 leaf (g: control; h: stressed with 150 mMNaCl for 48 h) and Pokkali leaf (i: control; j: stressed with 150 mMNaCl for 48 h), and OsVHA in rice cv. BRRI Dhan29 leaf (k: control; l: stressed with150 mM NaCl for 6 h) and Pokkali leaf (m: control; n: stressed with 150 mM NaCl for 1 h). Abbreviations: bs, bundle sheath; bu, bulliform cell; ep,epidermis; me, mesophyll; ph, phloem; xy, xylem. The specific signal is shown by green to yellow fluorescence of Alexa Fluor 488–5-dUTP (MolecularProbes, Leiden, the Netherlands), as indicated by the white arrow. The negative background and the negative control (o) are shown in red.



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On the other hand, in Pokkali the transcript of OsHKT1remained unaffected up to 6 h after NaCl stress in both theroot and shoot tissues and displayed a down-regulation48 h and 72 h after the stress. There was, however, someinduction of OsHKT1 in both the root and shoot tissues ofPokkali 24 h after the stress. As shown before, OsHKT1mediates Na+ influx, but not K+ influx (Horie et al., 2001).It has also been shown that OsHKT1 specifically mediatesNa+ uptake in rice roots when the plants are K+ deficient(Garciadeblas et al., 2003), and is induced by low-K+

conditions (Horie et al., 2001). In the present study, theinduction of OsHKT1 in root epidermis and vascularcylinder cells, as well as in shoot mesophyll cells of salt-sensitive BRRI Dhan29, might indicate its involvement inNa+ uptake by the root and in the subsequent circulation ofNa+ in the leaf mesophyll cells, where Na+ causes damage.

Since the experimental plants were grown with an optimalK+ concentration in the growth medium, there should be noK+ deficiency in cells under control conditions. However,under high NaCl conditions, Na+ competition at K+ bindingsites may result in K+ deficiency (Maathuis and Amtmann,1999), and thus might cause the induction of OsHKT1in both the cultivars observed here. Another possibility isthat under high NaCl conditions, excess Na+ entering thecytosol increases the optimal cytosolic Na+/ K+ ratio, whichcells might recognize as a K+ deficiency, thus inducingOsHKT1 as suggested by Horie et al. (2001) in cases ofK+ deficiency. The higher uptake of Na+ into the cytosol ofcv. BRRI Dhan29 than into that cv. Pokkali (Kader andLindberg, 2005) is probably caused by a faster inductionof OsHKT1 in BRRI Dhan29 (1 h and 6 h after the stress)than in Pokkali (24 h after the stress).With a longer stress period, the salt-tolerant cv. Pokkali

starts to down-regulate OsHKT1 expression in bothroots and shoots, as found here. The down-regulation ofOsHKT1 in leaf mesophyll cells in Pokkali explains thesalt-tolerance of this cultivar, possibly by hindering Na+-influx into these metabolically very important cells. Thedown-regulation of OsHKT1 in response to salt stress wasalso demonstrated by Horie et al. (2001) and Golldack et al.(2002a). However, the initial induction of OsHKT1 in thesalt-tolerant cv. Pokkali, for whatever reason, countersits salt-tolerance ability. Possibly, some post-transcriptionalchanges, or any conformational changes of the protein,hinder Na+ transport into Pokkali by means of this Na+

transporter. In a recent study, it was shown that the uptakeof Na+ by K+-selective channels/transporters was not foundin this cultivar (Kader and Lindberg, 2005).This study also suggests the involvement of OsHKT2

in the salt-stress response, especially in the salt-tolerantcv. Pokkali. There was a substantial induction (some 15-fold higher than in the control) of OsHKT2 in shoot tissueof Pokkali and to a lesser extent in root tissue of the samecultivar, but not in the salt-sensitive cv. BRRI Dhan29.Although OsHKT2 (a K+-Na+ coupled transporter) doesnot mediate K+ influx from a high K+ solution in theabsence of Na+, it does confer tolerance of salt stress underhigh Na+ conditions, probably by an increased K+ uptakeability, as demonstrated in Saccharomyces cerevisiae(Horie et al., 2001). In the present study, the induction ofOsHKT2 in the epidermis, exodermis, and xylem tissueof roots might indicate its involvement in K+ uptake andtransport through xylem. The induction in the mesophyllcells and in the transition from the phloem to mesophyllcells may indicate its involvement in the recirculation ofK+ within the mesophyll cells through the phloem. In addi-tion to metabolites such as sugars, minerals and salts alsocan use the phloem pathway to be redistributed from oldsource leaves towards young and expanding sink leaves(Sondergaard et al., 2004). Thus, the induction of OsHKT2in the salt-tolerant cv. Pokkali might confer salt tolerance

Fig. 6. Changes in the vacuolar area of shoot protoplasts of rice cvsBRRI Dhan29 and Pokkali upon salt stress (150 mM NaCl). Protoplastswere loaded with the vacuole-specific dye 6-CFDA. The dye in thevacuole was excited sequentially by the 458 nm and 488 nm lines of anargon-ion-laser, and the images of the vacuoles with the signal from thedye were detected in the 500–530 nm range after passing through a LeicaRSP500 short-pass filter (Leica). The images of the vacuoles were takenboth before and after salt addition. The area of the vacuole was measuredusing the Leica Confocal Software LCS Lite. Each column indicates theaverage vacuolar area as measured in 40 protoplasts. The data representmean values 6SE. Salt 1: protoplasts were stressed with NaCl 100 mMfor 5–10 min; Salt 2: protoplasts were stressed with 100 mM NaClfor 24 h.

Fig. 7. Calculated FRET-efficiencies of VHA-subunits. The FRET effectof the FRET-pairs VHA-a-YFP/VHA-c-CFP was measured in more than60 protoplasts. The FRET efficiency was calculated for each measure-ment. The data represent mean values 6SE.



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by increasing its expression in leaf tissue, through con-tributing to a low cytosolic Na+/K+ ratio, as suggested byHorie et al. (2001). Maathuis (2006) recently reporteda greater than 3-fold change of Na+-K+ symport in responseto salt stress. Several other K+-transporter genes have alsobeen shown to be up-regulated under high NaCl conditions,which possibly reflects a plant’s ability to maintain specificcytosolic K+ levels at various capacities. Salt stress in-creases the transcript of the K+-transporter genes AtKC1(Pilot et al., 2003), KMT1 and various HAK/KUP (Suet al., 2001, 2002). The ability to maintain ionic homeo-stasis was shown to be an important salt-tolerance deter-minant in barley when it was compared with a moderatesalt-sensitive rice cultivar IR64 (Ueda et al., 2006). Thisstudy also revealed that the regulatory mechanism forcontrolling K+/Na+ homeostasis in cells of the salt-tolerantcv. Pokkali seemed to work by increasing K+ uptake (byinducing the expression of OsHKT2), and then also byreducing Na+ influx (by decreasing the expression ofOsHKT1). These mechanisms were not as efficient in BRRIDhan29 as in Pokkali. Since K+, at a high concentration,also is inhibitory for enzymatic functions (Greenway andOsmond, 1972), the induction of OsHKT2 in Pokkalileaves decreased over the course of the stress period.In addition to the increased uptake of K+ and decreased

Na+-influx, as proposed, Pokkali might maintain K+/Na+

homeostasis in the cytosol by compartmentalizing cytosolicNa+ into the vacuole. Upon NaCl stress in Pokkali, rel-atively quick (1 h after the stress) induction of OsVHA wasfound in this cultivar. Although the expression of OsVHAwas induced in the salt-sensitive cv. BRRI Dhan29, theinduction was much delayed (until 6 h after the stress)and was less than that of the salt-tolerant cv. Pokkali. Thisresult is consistent with an earlier study, where we dem-onstrated the compartmentalization of Na+ into the vac-uole after few minutes of NaCl stress in Pokkali, but not inBRRI Dhan29 (Kader and Lindberg, 2005). The inducedexpression of OsVHA upon salt stress was found mainlyin the epidermis of the roots. This is quite rational, sincethese cells first encounter the excess of cytosolic Na+,before it enters into the xylem through the symplastic path-way to be transported into the shoot. The induction in thephloem and the transition from the phloem to mesophyllcells in leaves might also indicate the effort of these cellsto keep the harmful Na+ away from the mesophyll cells.In addition to maintaining cytosolic ionic homeostasis

and pH, VHA was also shown to be important for salttolerance in Saccharomyces cerevisiae (Hamilton et al.,2002) and in many plant species (Golldack and Dietz,2001; Kluge et al., 2003a; Senthilkumar et al., 2005;Vera-Estrella et al., 2005). Some variable expression ofOsVHA with time was observed under salt stress in bothcultivars studied (Fig. 3). This increase–decrease–increaseinduction of OsVHA in both the cultivars might indicatecertain limitations of the vacuole as a sink for cytosolic

Na+. Immediately after the stress, the salt-tolerant cv.Pokkali increased the expression of VHA; the inductioncontinued to increase until 6 h after the stress, and thuspossibly compartmentalized cytosolic Na+ into the vacuole.Thereafter, a high amount of Na+ in the vacuole might havereduced OsVHA induction, which decreased nearly to thecontrol level 48 h after the stress. At 72 h after the stressOsVHA is again induced, which might be correlated withcell growth.The FRET-measurements between two different subunits

of a particular protein fused with fluorophores, such as CFPand YFP, facilitate quantitative estimates of distance interms of the efficiency of resonance energy transfer (Seidelet al., 2005). Thus, the changes in FRET efficiency betweentwo different subunits of VHA upon salt stress might bea good indication of any structural change of the proteindue to salt stress. In general, salinity affects the expressionof VHA-genes in plants. One important mechanism of salt-tolerance in plants seems to be the adaptation of transportcapacities via changes in the amount of VHA and there-fore of VHA activity (Ratajczak, 2000; Wang et al., 2001;Kluge et al., 2003b). However, so far no report has dem-onstrated any structural change of VHA due to salt stress,though some ATP-hydrolysis and proton transport datado reveal changes in the coupling ratio of plant VHA uponexposure to salt stress (Ratajczak, 2000).The FRET efficiency between the proteolipid VHA-c and

the C-terminal transmembrane domain of VHA-a decreasedslightly in cv. Pokkali, but increased significantly in cv.BRRI Dhan29 upon NaCl stress. Signalling for the regu-lation of VHA is transduced from the V1 to V0 domain viaconformational changes in VHA-a (Landolt-Marticorenaet al., 1999). On the other hand, flexibility in the stoi-chiometry of the proteolipid VHA-c within the complexmight also explain the observed structural changes. Thesechanges could be due either to an increase in the diameter ofthe proteolipid-ring (Klink et al., 1990; Ratajczak, 2000) orto the replacement of VHA-c by isoforms, as indicated bydifferential immunological cross reactions (FischerSchliebset al., 1997). Changes in the coupling ratio of the V-ATPasefrom Mesembryanthemum crystallinum (Rockel et al.,1994) support this hypothesis. Although the analysedVHA subunits are derived from M. crystallinum, artificialeffects can be excluded, because the increase of FRETefficiency was specific to cv. BRRI Dhan29 upon salinityexposure. Nevertheless, the differential changes of FRETefficiency between VHA-a and VHA-c in Pokkali andBRRI Dhan29 might be correlated with their differentiallevels of Na+-compartmentalization into the vacuole.Under salt stress, both the increased vacuolar compart-

mentalization ability of Na+ (by inducing the expression ofVHA) and decreased uptake of Na+ into the cytosol (bydecreasing the expression of OsHKT1) seem to work moreefficiently in the salt-tolerant cv. Pokkali than in the salt-sensitive cv. BRRI Dhan29. Maathuis (2006) suggested



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that both the down-regulation of HKT1 and the up-regulation of an NHX isoform (tonoplast Na+/H+ anti-porter) could contribute greatly to limiting Na+ loading inplant tissue, particularly when cytosolic Na+ contents areconcerned.

Conclusion

With respect to OsHKT1, OsHKT2, and OsVHA expres-sion data, the regulatory mechanism of cytosolic Na+/K+

homeostasis seems to be an important salt-tolerance de-terminant in the salt-tolerant rice cv. Pokkali. This mech-anism is less efficient in the salt-sensitive cv. BRRIDhan29. At the onset of NaCl stress, Pokkali increasesthe expression of OsHKT2 in both the root and shoottissues. Since the induction is very strong in the shoot, inparticular in the mesophyll cells, the OsHKT2 possiblyenhances the recirculation of K+ in metabolically active leafmesophyll cells under high NaCl conditions, to maintain anadequate cytosolic Na+/K+ ratio. Although the cells alsotake up Na+ along with K+ by means of this transporter,cells might transport Na+ back from the mesophyll cellsby means of OsHKT8, as suggested by Rus et al. (2005)and as delineated in Fig. 8. As with OsHKT2, Pokkali alsoinduces the expression of OsVHA at the onset of high NaClconditions, most likely to compartmentalize cytosolic Na+

into the vacuole. The induction of OsVHA decreases nearly

to the control expression levels in root tissue 24 h afterthe stress and in shoot tissue 48 h after the stress, althoughOsVHA expression increases 72 h after the stress. More-over, Pokkali induces the expression of the Na+ transporterOsHKT1 24 h after NaCl stress. This might occur eitherbecause of K+ deficiency in cells (caused by Na+ com-petition at transport sites), or by interruption of thecytosolic Na+/K+ ratio, which cells might sense as a K+-deficiency. However, at a certain stage later on, Pokkalidown-regulates the expression of OsHKT1. It is concludedthat, at the onset of high NaCl conditions, Pokkali main-tains cytosolic Na+/K+ homeostasis by increasing theNa+-K+ coupled uptake through the induction of OsHKT2,as well as by increasing the compartmentalization ofcytosolic Na+ into the vacuole. The latter is facilitated bythe induced expression of OsVHA. There is, however, nostructural change of OsVHA, as estimated in terms ofthe FRET efficiency between VHA-a and VHA-c, in cv.Pokkali. Pokkali might also maintain a low influx of cyto-solic Na+, either by means of a conformational changeof the OsHKT1 protein and/or any post-transcriptionalchanges of the OsHKT1 gene. On the other hand, 48 h afterthe stress, Pokkali maintains cytosolic K+/Na+ homeostasisby down-regulating OsHKT1. However, to understand themechanism of K+/Na+ homeostasis in rice fully, the cell-and tissue-specific expression patterns of other membersof the HKT family need to be investigated under con-ditions of NaCl stress.

Fig. 8. A model describing possible functions of OsHKT1, OsHKT2, and OsVHA in regulating cytosolic Na+/K+ homeostasis. OsHKT1 (red ovals)functions as a Na+ uptake system (Garciadeblas et al., 2003), OsHKT2 (blue ovals) functions as a K+-Na+ coupled uptake system (Horie et al., 2001),OsHKT8 (green ovals) functions as a Na+ transporter (but plays an important role in maintaining K+ homeostasis in shoots; Rus et al., 2005), andOsVHA (yellow oval) energizes tonoplast Na+/H+ antiporter to compartmentalize cytosolic Na+ into vacuoles (Golldack and Dietz, 2001).Abbreviations: EC, epidermal cell; CC, cortical cell; EnC; endodermal cell; SC, stellar cell, VAC, vessel associated cell; BSC, bundle sheath cell; MC,mesophyll cell; CoC, companion cell.



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Acknowledgements

We gratefully acknowledge financial support from the IslamicDevelopment Bank (IDB) and from the Lennart Hjelm’s foundation,SLU. We thank Dr Vladislav V Yemelianov for his help to convertthe figures as TIFF file.

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