nacl-induced changes of ion fluxes in roots of transgenic bacillus thuringiensis (bt) cotton...

Journal of Integrative Agriculture2013, 12(3): 436-444 March 2013

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.doi:10.1016/S2095-3119(13)60244-0

RESEARCH ARTICLE

NaCl-Induced Changes of Ion Fluxes in Roots of Transgenic Bacillusthuringiensis (Bt) Cotton (Gossypium hirsutum L.)

LI Mao-ying, LI Fang-jun, YUE Yue-sen, TIAN Xiao-li, LI Zhao-hu and DUAN Liu-sheng

State Key Laboratory of Plant Physiology and Biochemistry /Engineering Research Center of Plant Growth Regulator,Ministry of Education/College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, P.R.China

Abstract

Bacillus thuringiensis (Bt) cotton is grown worldwide, including in saline soils, but the effect of salinity on ion fluxes ofBt cotton remains unknown. Responses of two transgenic Bt cotton genotypes (SGK321 and 29317) and their correspondingreceptors, Shiyuan 321 (SY321) and Jihe 321 (J321), to 150 mmol L-1 NaCl stress were studied in a growth chamber. The rootdry weight of SGK321 and 29317 under NaCl treatment was decreased by 30 and 31%, respectively. However, theircorresponding receptor cultivars SY321 and J321 were less affected (19 and 24%, respectively). The root length andsurface area of the Bt cultivars were significantly decreased relative to their receptors under salt stress. NaCl treatmentsignificantly increased Cry1Ac mRNA transcript levels in SGK321 and 29317 but did not affect Bt protein content in leavesor roots of either cultivar at 1 and 7 d after NaCl treatment. Fluxes of Na+, K+, and H+ in roots were investigated using thescanning ion-selective electrode technique. Both mean K+ efflux rate and transient K+ efflux of the Bt cultivars increasedfour-fold compared to their corresponding receptors when exposed to salinity stress. There were no significant differencesin Na+ efflux between Bt and non-Bt cottons. Furthermore, the Na+ contents in roots and leaves of all genotypesdramatically increased under salt stress, whereas K+ contents decreased. Our results suggested that Bt cotton cultivarsare more sensitive to salt stress than their receptor genotypes.

Key words: transgenic Bt cotton, salinity stress, toxin protein, SIET, K+ flux

INTRODUCTION

More than 800 million ha of land worldwide, takingover 6% of the world’s total land area, are affected bysalt stress (Munns and Tester 2008). Most of the saltstress has arisen from natural causes, such as the ac-cumulation of salts over long periods of time in aridand semiarid zones (Rengasamy 2010). There are 36million ha of saline lands in China, accounting for4.88% of the total available lands. In addition, 9.2 mil-lion ha of cultivated lands, 6.62% of the total, have

become salinized (Yang 2008) and are restricted to salt-tolerant crops.

Cotton is classified as moderately salt-resistant (Leidiand Saiz 1997). Transgenic cotton expressing Bacil-lus thuringiensis (Bt) toxins provides highly effectivecontrol of cotton bollworm. These crops have sub-stantially increased yields, used fewer conventionalchemical pesticides, and caused less environmental pol-lution (Jiang et al. 2006; Luo et al. 2008; Chen et al.2012). They are currently cultivated on a large com-mercial scale in many countries, including the UnitedStates of America (Adamczyk and Meredith 2004),

Received 23 February, 2012 Accepted 28 May, 2012Correspondence DUAN Liu-sheng, Tel/Fax: +86-10-62731301, E-mail: [email protected]

NaCl-Induced Changes of Ion Fluxes in Roots of Transgenic Bacillus thuringiensis (Bt) Cotton (Gossypium hirsutum L.) 437

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.

Australia (Whitehouse et al. 2005), and China (Luo et al.2008; Chen et al. 2012). The efficacy of transgenic Btcotton closely correlates to endotoxin expression levels.A number of researchers have reported variability inefficacy of Bt cotton with plant age (Wan et al. 2005)and structure (Abel and Adamczyk 2004), or under cer-tain biotic and abiotic stresses (Mahon et al. 2002; Chenet al. 2005; Xia et al. 2005; Jiang et al. 2006; Dong et al.2007; Chen et al. 2012). There were also some re-ports that the introduction of foreign genes (Bt Cry1Ac)might affect the metabolism of transgenic cotton. Forexample, Bt cotton has a lower K+ efficiency, making itmore sensitive to K+ deficiency than non-transgeniccotton cultivars (Yang et al. 2011).

Extensive research has focused on the effects ofsalinity on both transgenic and conventional cotton cul-tivars (Ashraf et al. 2002; Jiang et al. 2006). Salinitystress has negative effects on the growth and photo-synthetic capacity of cotton (Meloni et al. 2003; Konget al. 2011). In recent years, the non-invasive scan-ning ion-selective electrode technique (SIET) has be-come important for its ability to measure ion fluxes andelucidate the dynamic changes in ion relations causedby salinity (Shabala and Cuin 2008; Sun et al. 2009a).The importance of the transient and dynamic flux pro-files for clarifying ion transport mechanisms in plantsare widely accepted (Chen et al. 2005). Clearly, K+

homeostasis plays a crucial role in the salt adaptation ofplant cells. In some reports, the correlation betweenK+ efflux and whole-plant responses is very strong(Shabala and Cuin 2008).

In this study, two transgenic cotton cultivars and theircorresponding receptor cotton cultivars were treated at

the seedling stage with 150 mmol L-1 NaCl in nutrient so-lution and then examined using SIET. The objectives wereto determine the effects of NaCl salinity stress on seedlinggrowth and ion relations of two transgenic Bt cotton cul-tivars and their corresponding receptor cotton cultivars.

RESULTS

Salt stress inhibits the plant growth

After 150 mmol L-1 NaCl treatment for 7 d, the root dryweight of SGK321 and 29317 under NaCl salinity de-creased by 30 and 31%, respectively, compared to theuntreated control. However, their corresponding re-ceptor cultivars SY321 and J321 were less affected(19 and 24%, respectively). The root/shoot ratios ofSY321, SGK321, and J321 were much higher than no-stress control, but that of 29317 was lower than control.Salinity treatment had a strong impact on total root lengthand total root surface area. The total root lengths ofSGK321, 29317, and SY321 decreased 26, 30, and 36%,respectively, relative to the control and that of J321decreased only 2%. Total root surface area decreasedabout 9-24% in all cultivars compared with their corre-sponding control (Table 1). However, the average rootdiameter showed no statistically significant reduction(data not shown).

Insecticide toxin proteins in Bt cotton

Leaf Bt protein contents were not affected for SGK321and 29317 at 1 and 7 d after salt stress (Fig. 1-A). And

Table 1 Effects of NaCl on seedling root dry weight (RDW), shoot dry weight (SDW), root/shoot ratio (R=RDW/SDW), total root length(TRL), and total root surface area (TRSA) of two transgenic cotton cultivars (SGK321 and 29317) and their corresponding receptor cottoncultivars (SY321 and J321)

NaCl (mmol L-1) Cultivars RDW (mg) SDW (mg) R TRL (cm) TRSA (cm2)0 29317 35.80 d 171.70 c 0.21 b 468.87 d 56.76 d

J321 60.20 ab 270.00 a 0.22 b 650.36 b 98.07 bSGK321 62.50 ab 296.20 ab 0.21 b 710.26 b 96.74 b

SY321 69.20 a 309.20 a 0.22 b 866.63 a 122.54 a150 29317 24.80 e 116.30 d 0.15 c 328.45 e 48.22 d

J321 46.00 cd 158.00 c 0.29 a 637.29 b 89.46 bSGK321 43.80 d 156.30 c 0.28 a 524.96 cd 76.43 c

SY321 56.20 bc 187.30 c 0.30 a 558.70 b 92.60 bCultivars (C) 0.0001 0.0001 0.0001 0.0001 0.0001NaCl 0.0001 0.0001 0.0037 0.0001 0.0001C×NaCl 0.7606 0.0037 0.0004 0.0001 0.0147

Values in the same column followed by the same letters are not significantly different at P=0.05, as determined by Duncan’s multiple range tests. The same as below.

438 LI Mao-ying et al.


root Bt protein contents for SGK321 and 29317 withsalt treatment also had a similar trend (data not shown).However, the Bt protein concentrations of SGK321 were

much higher than 29317.The cotton UBQ7 gene amplification was used as

an estimate of total RNA concentration across allsamples of both cultivars. The relative amount ofCry1Ac mRNA transcript in leaves peaked after 24 h ofNaCl treatment in both transgenic Bt cotton cultivars;the mRNA transcript levels Cry1Ac in cultivar SGK321increased 13-fold over the control group, while thoseof 29317 increased 6-fold (Fig. 1-B). After both 12and 48 h of salt stress, the Bt mRNA levels in leaves ofSGK321 were significantly higher than that of thecontrol, but there were no significant changes in tran-script levels in 29317 at these times. Semi-quantitativeRT-PCR yielded similar results (Fig. 2).

Sodium and potassiumcontents in plant tissues

The sodium content of roots and leaves of all cultivarsincreased dramatically, by 7- to 14-fold, after NaCl treat-ment (Table 2). The contents of potassium decreasedsignificantly in all cultivars. The Na+/K+ ratio in planttissue is a key index presenting plant tolerance to salt.Salt treatment caused the ratio to increase in both rootsand leaves of all cultivars. The Na+/K + ratio of leaf androot for 29317 were significantly higher than J321 un-der salt stress.

Ion flux responses to salt stress

NaCl treatment caused dramatic changes in net Na+,K+, and H+ fluxes on the cotton root surface. It causedsignificantly increased effluxes of Na+ and K+ as wellas a significantly decreased efflux of H+. The salt stresscaused a net K+ efflux, ranging from 128 to 694 pmolcm-2 s-1 measured in the root tip. A vigorous NaCl-induced K+ efflux was found in 29317 (Fig. 3-A) and

Fig. 1 Effects of NaCl on Bt protein concentrations (A) and Btgene transcript levels (B) in leaves of two transgenic cottoncultivars. Data represent means of three replicates±SD. Allreplicates yielded similar results.

Fig. 2 Semi-quantitative RT-PCR analysis of Bt Cry1Ac in response to NaCl stress treatment in leaves of two transgenic cotton cultivars.



SGK321 (Fig. 3-C). Their mean flux rates increased5-fold compared to their corresponding receptor culti-vars J321 (Fig. 3-B) and SY321 (Fig. 3-D).

Transient K+ flux responses to salinity treatment werealso detected in our experiment. The K+ efflux exhib-ited a two-phase response. There was instantaneousK+ efflux increase of more than 1 000 pmol cm-2 s-1 inthe root tips of all cultivars. When the K+ efflux stabi-lized after about 10 min of NaCl treatment, SGK321had a significantly higher K+ efflux than its receptorcultivar, SY321 (Fig. 4-B). This difference was notseen between 29317 and J321 (Fig. 4-A).

Salt stress also induced a dramatic Na+ efflux in allcultivars. Relative to the controls, the mean Na+ fluxrate increased 4-fold in SY321 and SGK321, 4.5-foldin J321, and 6.5-fold in 29317 after 24 h NaCl treatment.However, Na+ fluxes in Bt and non-Bt cotton did notsignificantly different (Fig. 5-A).

NaCl induced a marked decrease in the H+ efflux inall cultivars. Compared to the controls, the mean H+

efflux of SY321 and SGK321 decreased to 46 and28 pmol cm-2 s-1, the mean H+ flux of 29317 influxedvery slightly, and the mean H+ flux of J321 was alsoefflux (Fig. 5-B).

DISCUSSION

Plants differ greatly in their tolerances to salinity, asreflected in their different growth responses (Munnsand Tester 2008). Salt stress can reduce seeding growthof both transgenic and conventional cotton cultivars(Ashraf et al. 2002; Jiang et al. 2006), and our resultsshowed similar patterns to previous research. In general,

root biomass was more strongly affected than shootbiomass under salinity stress. The data displayed inTables 1 and 2 indicated that roots were more sensitivethan shoots to NaCl treatment. Except in the mostsensitive cultivar, 29317, the root/shoot ratios increased.The higher root/shoot ratio under NaCl treatment couldbe attributed to the outstanding ability of roots to resistsalt stress, allowing more root than leaf biomass undersalt-stress conditions (Munns and Tester 2008). Wefound that salinity treatment significantly reduced rootlength and root surface area. However, the averageroot diameter did not change statistically, indicating thatthe inhibiting effect on root growth was mainly throughreducing root length.

The levels of Bt toxin proteins are increasingly un-derstood to be affected by environmental stresses suchas nutrient and water deficiency and salinity. The ex-pression of other introduced genes in genetically modi-fied crops was also reported to be significantly affectedby environment stresses (Dong and Li 2007). Quanti-tative real-time polymerase chain reaction (qPCR)-basedmethods have been successfully employed to quantifyexpression levels of transgenes in plants carrying dif-ferent Bt toxins (Adamczyk et al. 2009). We found Btprotein concentrations of seedling leaves and roots werenot affected by short-term salt stress. Meanwhile, theamount of Cry1Ac mRNA transcripts in leaves of bothtransgenic cultivars increased significantly after NaCltreatment. These results may be due to protein transla-tion occurring after RNA transcription.

Whether plants can survive excess salinity dependsto a large extent on their abilities to maintain ionic ho-meostasis under saline conditions. Maintaining low Na+

and Cl- levels while keeping high concentrations of nu-

Table 2 Effects of NaCl on K+ and Na+ concentrations and Na+/K+ ratios of seedling roots and leaves of two transgenic cotton cultivars(SGK321 and 29317) and their corresponding receptor cotton cultivars (SY321 and J321)

NaCl (mmol L-1) Cultivars K+ (mg g-1) Na+ (mg g-1) Na+/K+ ratiosRoots Leaves Roots Leaves Roots Leaves

0 29317 48.69 bc 18.54 ab 9.35 e 6.42 d 0.19 c 0.35 dJ321 46.74 cd 19.13 a 8.42 e 6.99 d 0.18 c 0.36 d

SGK321 50.65 ab 17.95 b 8.78 e 8.25 d 0.17 d 0.46 dSY321 52.44 a 18.16 b 8.62 e 7.37 d 0.16 d 0.41 d

150 29317 39.90 e 11.12 c 87.89 a 98.56 b 2.21 a 8.87 bJ321 38.64 e 11.09 c 66.01 d 73.37 c 1.71 d 6.67 c

SGK321 39.85 e 10.95 c 83.03 b 74.78 c 2.08 b 6.83 cSY321 44.57 d 10.19 c 74.27 c 104.13 a 1.67 d 10.22 a

Cultivars (C) 0.0001 0.0252 0.0001 0.0001 0.0001 0.0001NaCl 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001C×NaCl 0.0687 0.2846 0.0001 0.0001 0.0001 0.0001



Fig. 3 Net K+ fluxes (positive influx) of control and NaCl-treated seedling roots of two transgenic cotton cultivars (29317 and SGK321)and their corresponding receptor cotton cultivars (J321 and SY321). A, 29317. B, J321. C, SGK321. D, SY321. Seedlings at the one-leafstage were subjected to 150 mmol L-1 NaCl solution for 24 h. Young roots tips 2-3 cm in length were sampled from four cultivars and usedfor flux K+ measurements. The same as below. Each point represents the mean of 6-8 seedlings.



trient elements like K+ is crucial for plant salt tolerance(Zhu 2001; Munns and Tester 2008). The ability ofplant cells to maintain an optimal K+/Na+ ratio in thecytosol enables normal cell metabolism (Chen et al.2005). Our results showed that Bt cotton maintained alower potassium content and a higher sodium contentthan their receptor cotton cultivars (Table 2). We foundthat the transgenic Bt cotton cultivars, SGK321 and29317, were more sensitive to NaCl treatment than thenon-transgenic cultivars, SY321 and J321.

The capacity of plants to counteract salinity stressstrongly depends on their potassium nutritional status(Maathuis 2009). Thus, the ability of plant cells toretain K+ is more crucial for salt tolerance than theirability to restrict Na+ uptake (Chen et al. 2005). Wefound that NaCl treatment caused dramatic changes in

net K+ flux from cotton root surfaces. The mean K+

flux rate increased Bt cultivars 5-fold more than theircorresponding receptor cultivars. Transgenic cottonexpressing Bt toxins are currently cultivated on a largecommercial scale in many countries. The Bt endotoxinhydrolyzes an inhibitor of potassium transport, presum-ably a polypeptide, in the midguts of Lepidoptera (Yanget al. 2011). Dramatic change in the net K+ flux oftransgenic Bt cotton may be due to the introduction offoreign genes (Bt Cry1Ac) encoding insecticidal pro-tein into plants.

Na+ uptake, transport, and compartmentalization arecrucial for plants to survive saline environments. NaClcan induced a dramatic Na+ efflux in all cultivars,however, Na+ flux in Bt and non-Bt cottons did notsignificantly differ (Fig. 5-A). Unfortunately, measure-

Fig. 4 Transient K+ kinetics of salt shock (150 mmol L-1 NaCl) measured on seedling roots of two transgenic cotton cultivars (29317 andSGK321) and their corresponding receptor cotton cultivars (J321 and SY321). A, fluxes for 29317 and J321. B, fluxes for SGK321 andSY321. Seedlings at the 3rd-leaf stage were used for the transient ion flux kinetics. Prior to the salt shock, steady state K+ fluxes wereexamined. Each point represents the mean of six seedlings.



ments of Na+ and Cl- were complicated by the low sig-nal-to-noise ratio on the liquid ion exchanger used atsuch high external ion concentrations (Chen et al. 2005;Sun et al. 2009a), and this limited the use of the Na+

flux. H+ fluxes offer direct evidence of ion exchangecoupling with H+, although there are several types ofsecondary transport systems located at the plasmamembrane, e.g., Na+/H+ antiporters, H+/Cl- symporters,and H+/K+ symporters. Kong et al. (2011) reportedthat the Na+ extrusion in salt-stressed cotton roots was

mainly attributed to an active Na+/H+ antiporter acrossthe plasma membrane. Other workers have reportedspecies-specific differences in H+ flux upon salt stress(Zhu 2002; Shabala and Cuin 2008; Sun et al. 2009b).

CONCLUSION

In summary, the fresh (data not show) and dry weightsof roots and shoots, root length, and total root superfi-cial area all significantly decreased under NaCl treatment.The Na+ content of roots and leaves for all cultivarsdramatically increased, and K+ decreased after NaCltreatments. The mean K+ efflux rate of Bt cotton culti-vars SGK321 and 29317 increased significantly com-pared with their corresponding receptor cultivars,SY321 and J321, under salinity stress, suggesting thatBt cottons are more sensitive than their receptor cottoncultivars to salt stress. Further studies will be neces-sary to investigate the effects of endogenously-producedBt endotoxins on the potassium efficiencies of Btcottons.

MATERIALS AND METHODS

Plant materials

We used two transgenic cotton cultivars for this study: (1)SGK321 was developed at the Shijiazhuang Academy ofAgricultural Sciences, Hebei Province, China, by introduc-ing foreign Bt Cry1Ac genes into the conventional cottoncultivar Shiyuan 321 (SY321); and (2) line 29317 was devel-oped by Shanxi Academy of Agricultural Sciences,Yuncheng, China, by introducing foreign Bt Cry1Ac genesinto the conventional cotton cultivar Jihe 321 (J321). Twoconventional upland cotton cultivars SY321 and J321 wereused as a control. All cultivars were obtained from cottonbreeding institutes or their associated seed companies.

Growth conditions and treatment

The research was conducted in a growth chamber under28/20°C, 14/10 h day/night, and 450 �mol m-2 s-1 lightconditions. Seeds were surface sterilized and germinated.When the two cotyledons were just fully expanded, seed-lings were carefully transferred into tap water for 1 d. Theseedlings were cultured hydroponically for 4 d in Hoaglandsolution, and then they were subjected to salt stress byadding 150 mmol L-1 NaCl to the Hoagland solution. Deion-

Fig. 5 Effects of NaCl on the mean fluxes of Na+ and H+ fluxes(positive influx) measured on seedling roots of two transgenic cottoncultivars (29317 and SGK321) and their corresponding receptorcotton cultivars (J321 and SY321). A, mean Na+ fluxes for 29317,J321, SGK321, and SY321. B, mean H+ fluxes for 29317, J321,SGK321, and SY321. Seedlings at the 1st-leaf stage were subjectedto 150 mmol L-1 NaCl solution for 24 h. Young roots tips 2-3 cmin length were sampled from four cultivars and used for flux Na+ andH+ measurements. All experiments were repeated six times withsimilar results. Different letters above the bars indicate statisticallysignificant differences among cultivars.



ized water was added daily to replace water lost bytranspiration. After 7 d of NaCl treatment, sodium and po-tassium contents, seedling growth, and Bt protein concen-tration were measured. For ion flux experiments, seedlingsat the 1st-leaf stage were subjected to 150 mmol L-1 NaClsolution for 24 h. Young roots tips 2-3 cm in length weresampled from four cultivars and used for flux Na+, K+, andH+ measurements. And seedlings at the 3rd-leaf stage wereused for the transient K+ ion flux kinetics.

Analysis of root traits

Whole roots were scanned with an EPSON TransparencyUnit (Seiko Epson Corp., Tokyo, Japan). Total root lengthand total root surface area were determined with WinRHIZOsoftware (ver. 4.0b, Regent Instruments Inc., Quebec,Canada).

Bt protein concentrations

Bt protein contents were determined using a commercialquantification kit (EnviroLogic, Portland, ME, USA).Samples of the first fully-expanded true leaves and rootswere weighed to accurately determine the amount of start-ing material. The samples were combined in a 5-mLmicrocentrifuge tube and homogenized by hand in Cry1Acextraction buffer using a fitted pestle. This sandwich en-zyme-linked immunosorbent assay (ELISA) uses a colordevelopment step where the intensity of color is propor-tional to Cry1Ac protein content in the sample extract. TheCry1Ac protein was quantified spectrophotometrically incomparison to a standard curve based on Cry1Ac calibra-tors supplied in the kit. The amount of Cry1Ac was ex-pressed as ng g-1 FW.

Bt gene transcript level

Total RNA were extracted from frozen samples (12, 24, and48 h control and NaCl-treated samples) using an RNA ex-traction kit (BioTeke Corporation, Beijing, China), andcDNA was synthesized from total RNA by using a BioTekeSuper RT Kit (BioTeke Co., Beijing, China). Semi-quantita-tive RT-PCR was carried out using the UBQ7 gene of cot-ton as an endogenous reference to determine the relativequantities of the target transcripts. The primers used forr e a l - t i m e P C R w e r e U B Q 7 f o r w a r d ( 5 ´-GAAGGCATTCCACCTGACCAAC-3´) and reverse (5 -́CTTGACCTTCTTCTTCTTGTGCTTG-3´) primers andCry1Ac forward (5 -́CGCGAGGAAATGCGTATTCAAT-3´)and reverse (5 -́ACAATGGGATAGCTGTGGTCAAG-3´)primers. Real-time QRT-PCR using SYBR Green II (TaKaRa,Dalian, China) was carried out in an ABI PRISM 7500 FastReal-time PCR System (Applied Biosystems, China). Real-

time PCR was performed in 20 �L-reactions containing0.4 �L ROX Reference Dye II, 2 �L 5-fold diluted-cDNA,0.4 �L of 10 �mol L-1 each primer, and 10 �L SYBR GreenPCR Master Mix (TaKaRa, Dalian, China). The mean val-ues from three replicates were normalized against UBQ7.All experiments were repeated at least three times with simi-lar results.

Measurement of sodium and potassium contents

Seedlings were separated into roots and shoots, then oven-dried at 80°C for 72 h and weighed. After weighing, the drysamples were ground to a fine powder, screened through a0.5-mm sieve, soaked in 1.0 mol L-1 HCl for 24 h, shaken for30 min, and filtered. Filtered solutions were analyzed forsodium and potassium by atomic-absorption spectroscopy(SpectAA-50/55, Varian, Australia).

Measurement of K+, Na+, and H+ flux

Net fluxes of K+, Na+, and H+ were measured non-invasivelyby Xuyue Science & Technology Co. (Beijing, China)(http://www.xuyue.net) with the BIO-IM Non-InvasiveMicro-test System (YoungerUSA, Amherst, MA, USA).The concentration gradients of the target ions were mea-sured by moving the ion-selective microelectrode betweentwo positions close to the plant materials. The electrodemoved from one position to another in a predefined sam-pling routine while also being scanned with the three-di-mensional micro-stepper motor manipulator (CMC-4). Ion-selective electrodes of the following target ions were cali-brated prior to flux measurements: Na+, 0.5 and 5.0 mmol L-1

NaCl; H+, pH 5.5, 6.0, 6.5; K+, 0.05 and 0.5 mmol L-1 KCl. Themeasuring site, where vigorous fluxes of Na+, K+, and H+

were usually observed, was 30 �m from the root apex.Steady-state ion fluxes were measured for 10 min, the NaCltreatment was applied and the transient ion flux kineticswas measured for a further 10-20 min after treatment. Datarecorded during the 1st min after treatment was discardedbecause of diffusion effects from the addition of the stock.The flux data were recorded with MageFlux developed bythe Xuyue Company (http://xuyue.net/mageflux; Sun et al.2009b).

Statistics analysis

Data were statistically analyzed with the DPS (Tang andFeng 1997). Means were separated using Duncan’s mul-tiple range test at significance levels of 0.05 or 0.01.

AcknowledgementsThis work was supported by the National Natural ScienceFoundation of China (30871490) and the Specialized Re-



search Fund for the Doctoral Program of Higher Educationof China, and the Innovation Fund for Graduate Studentsof China Agricultural University (KYCX2011007).

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