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Journal of Plant Physiology 170 (2013) 355– 359

Contents lists available at SciVerse ScienceDirect

Journal of Plant Physiology

j o ur nal homepage: www.elsev ier .com/ locate / jp lph

hort communication

on-invasive microelectrode cadmium flux measurements reveal the spatialharacteristics and real-time kinetics of cadmium transport in hyperaccumulatornd nonhyperaccumulator ecotypes of Sedum alfredii

ian Suna, Ruigang Wangb,c,∗, Zhongqi Liub,c, Yongzhen Dingb,c, Tingqiang Lid

College of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, PR ChinaCenter for Research in Ecotoxicology and Environmental Remediation, Agro-environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, PR ChinaOpen Key Laboratory of Agro-environment and Food Safety of the Ministry of Agriculture, Tianjin 300191, PR ChinaMinistry of Education Key Laboratory of Environmental Remediation and Ecological Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058,R China

r t i c l e i n f o

rticle history:eceived 17 May 2012eceived in revised form 16 October 2012ccepted 17 October 2012

eywords:edum alfrediiadmium flux

a b s t r a c t

This study aims to determine the spatial characteristics and real-time kinetics of cadmium transport inhyperaccumulator (HE) and nonhyperaccumulator (NHE) ecotypes of Sedum alfredii using a non-invasiveCd-selective microelectrode. Compared with the NHE S. alfredii, the HE S. alfredii showed a higher Cdinflux in the root apical region and root hair cells, as well as a significantly higher Cd efflux in the leafpetiole after root pre-treatment with cadmium chloride (CdCl2). Thus, HE S. alfredii has a higher capabilityfor the translocation of absorbed Cd to the shoot. Moreover, the mesophyll tissues, isolated mesophyllprotoplasts, and intact vacuoles from HE S. alfredii exhibited an instantaneous influx of Cd in response to

oot hair cellntact vacuoleoot-to-shoot translocation

CdCl2 treatment with mean rates that are markedly higher than those from NHE S. alfredii. Therefore, thehyper-accumulating trait of HE S. alfredii is characterized by the rapid Cd uptake in specific root regions,including the apical region and root hair cells, as well as by the rapid root-to-shoot translocation and thehighly efficient Cd-permeable transport system in the plasma membrane and mesophyll cell tonoplast.We suggest that the non-invasive Cd-selective microelectrode is an excellent method with a high degree

e stu

of spatial resolution for th

ntroduction

Cadmium (Cd), a non-essential heavy metal widely presentsn the environment, is a pollutant that is highly toxic to alliving cells (Pilon-Smits, 2005; Mohanty et al., 2010). Previoustudies suggested that phytoremediation using hyper accumu-ator plants that can grow in contaminated soils is a usefulechnique to reduce or remove soil Cd contamination. The hyper-ccumulator plants reduce the soil Cd content by translocationnd accumulating high concentrations of the heavy metal in

heir shoots (Pilon-Smits, 2005; Mohanty et al., 2010). In the lastecade, several plant species were identified as Cd hyperaccumu-

ators, including Noccaea (Thlaspi) caerulescens, Arabidopsis halleri,

Abbreviations: Cd, cadmium; HE, hyper accumulator ecotype of Sedum alfredii;HE, non hyper accumulator ecotype of S. alfredii; PM, plasma membrane; SIET,

canning ion-selective electrode technique.∗ Corresponding author at: Center for Research in Ecotoxicology and Environmen-

al Remediation, Agro-environmental Protection Institute, Ministry of Agriculture,ianjin 300191, PR China. Tel.: +86 22 23612822; fax: +86 22 23612823.

E-mail address: 3761520835@sina.com (R. Wang).

176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jplph.2012.10.014

dy of Cd transport at the tissue, cellular, and sub-cellular levels in plants.© 2012 Elsevier GmbH. All rights reserved.

Thlaspi praecox, and Sedum alfredii. Since then, significant progresshas been made in understanding the physiological and molec-ular mechanisms of Cd hyperaccumulation (Verbruggen et al.,2009).

The progress in this field depends on the development of relatedtechniques to analyze Cd transport in plants. Of these methods,radiotracer 109Cd experiments, X-ray microfluorescence imaging,and Cd-sensitive fluorescent probe imaging have been widelyadopted and proven effective in the study of the flux and in vivolocalization of Cd in plants (Lu et al., 2008; Lu, 2008; Tian et al.,2011). Recent studies utilized the non-invasive measurement of ionfluxes to elucidate the dynamic changes in ion relations induced byenvironmental stresses. To date, two typical but similar techniquesdesigned to measure ion fluxes across membranes have been devel-oped, namely, the scanning ion-selective electrode technique (SIET)and the microelectrode ion flux estimation (MIFE) technique. SIEThas been used to characterize the Cd flux in higher plants (Pineroset al., 1998; Ma et al., 2010; He et al., 2011; Li et al., 2012). How-

ever, its application is limited to the roots (Pineros et al., 1998;He et al., 2011; Li et al., 2012) or suspension cells (Ma et al., 2010).Thus, SIET has not been fully applied in the monitoring of the spatialcharacteristics of Cd transport.

356 J. Sun et al. / Journal of Plant Physiology 170 (2013) 355– 359

Fig. 1. Effect of CdCl2 (10 �M for 2 h) pre-exposure on the net Cd fluxes in different regions of the roots in hyperaccumulator (HE) and nonhyperaccumulator (NHE) Sedumalfredii. (a) Representative images showing the measuring positions and Cd2+ flux profile in different regions, namely, the meristematic zone (region A), elongation zone(region B), mature zone (region C), and root hair cells (region D). The position and magnitude of the fluxes are indicated by arrows (black for HE and white for NHE); thearrows directed toward the root indicate influx, whereas those directed away from the root denote efflux and (b) Mean rates of Cd fluxes in the different regions. Each columnr o rootm

ril2wHmbpidt

M

P

wpuarwr

epresents the mean of four individual plants. At least two roots in each plant or twean. Columns labeled with a and b indicate a significant difference at P < 0.05.

The Cd/zinc (Zn) hyperaccumulating plant species S. alfrediiemarkably tolerates and hyperaccumulates Cd in its leaves. Thus,t is a useful model for the study of Cd transport and hyperaccumu-ation in plants (Yang et al., 2004; Lu et al., 2008, 2009, 2010; Lu,008; Tian et al., 2011). In this study, a Cd-selective microelectrodeas used to compare the Cd fluxes in the different cell types ofE and NHE S. alfredii, including the meristematic, elongation, andature zones of the roots, as well as the root hair cells, vascular

undles in the leaf petiole, mesophyll tissues, isolated mesophyllrotoplasts, and intact vacuoles. The results suggest that the non-

nvasive microelectrode is a highly effective tool that exhibits a highegree of spatial resolution in the study of the Cd transport at theissue, cellular, and sub-cellular levels in plants.

aterials and methods

lant materials

The spatial characteristics and real-time kinetics of Cd transportere investigated using Sedum alfredii, a Cd/Zn-hyperaccumulatinglant native to China. S. alfredii HE and NHE with healthy andniform shoots were selected and pre-cultured for 2 weeks in

basic nutrient solution for rooting according to a previouslyeported method (Yang et al., 2004). The pH of the nutrient solutionas adjusted daily to 5.5 using sodium hydroxide or hydrochlo-

ic acid. The plants were grown under greenhouse conditions at

hair cells in each root were measured. The bars represent the standard error of the

temperatures ranging from 20 ◦C to 25 ◦C and with a 16 h photope-riod and relative air humidity of 70%/85%. The nutrient solutionwas continuously aerated and renewed every 48 h. Seedlings withuniform roots (approximately 3–4 cm) were selected for the SIETexperiments.

Isolation of mesophyll protoplasts

Mesophyll protoplasts were isolated from the epidermis-removed leaves of the two ecotypes. The mesophyll tissueswere incubated in a cell wall-digesting medium consist-ing of a mannitol medium [500 mM mannitol, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 �M CaCl2, pH 5.5],1.5% cellulase Onozuka R-10 (Yakult Honsha), 1% cellulysin (Cal-Biochem), and 0.1% pectolyase Y-23 (Yakult Honsha) for 4 h at 28 ◦C.The protoplasts were then collected by filtration through a nylonmesh and further purified by gradient centrifugation (Cosio et al.,2004). The protoplasts were kept shortly in test tubes with ice priorto the SIET experiments.

SIET

Net fluxes of Cd were noninvasively measured using SIET (BIO-001A; Younger USA, LLC, MA, USA) (Sun et al., 2009, 2010; Maet al., 2010; He et al., 2011; Li et al., 2012). More details about thistechnique are provided in Supplemental method.

t Physiology 170 (2013) 355– 359 357

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Fig. 2. Transient Cd kinetics in the response of the roots to CdCl2 in HE and NHE S.alfredii. (a) Transient Cd kinetics. Each point represents the mean of five roots fromfive individual plants; the bars represent the standard error of the mean. The insetimages show the measuring point (500 �m from the root apex) and (b) The peakand mean rates of Cd flux during recording. Columns labeled with a and b represent

J. Sun et al. / Journal of Plan

xperimental protocols

teady-state Cd flux recordingThe roots of intact HE and NHE seedlings were exposed to a

0 �M CdCl2 for 2 h, rinsed with redistilled water, immediatelymmobilized in the measuring solution (10 �M CdCl2, 50 �M CaCl2,H 5.5), and then equilibrated for 10 min (Pineros et al., 1998).he steady Cd fluxes were then recorded in the meristematic zoneregion A), elongation zone (region B), mature zone (region C), andoot hair cells (region D). In each region, three or five points wereeasured for 2–3 min each (Fig. 1a).

ransient Cd flux recordingThe primary roots (which were measured 500 �m from the

oot apex where intense Cd flux usually occurs) of intact HE andHE seedlings, detached mesophyll tissues, and isolated mesophyllrotoplasts were immobilized in the basic solution (50 �M CaCl2,H 5.5; 500 mM mannitol was added to the protoplast measuringolution to maintain osmotic pressure) and then equilibrated for0 min. A steady-state Cd flux was then recorded for 10 min prioro the addition of 10 �M CdCl2. CdCl2 stock (200 �M) was slowlydded to the basic solution using a pipette to yield a final Cd con-entration of 10 �M. Afterward, Cd flux recording was restartednd then continued for 25–30 min. The data measured during therst 1–2 min was discarded because of the diffusion effects of stockddition. For transient Cd flux recording of intact vacuoles, theesophyll protoplasts were rapidly immersed in a hypo osmotic

olution for 3–5 min to release the intact vacuoles. The solutionas then replaced by a vacuolar measuring buffer (100 mM K-lu, 500 mM mannitol, 1 �M CaCl2, and 1 mM ATP, pH 7.3). After

he vacuoles were fixed on the bottom of the measuring cham-er (a 3.5 cm diameter polylysine-pretreated culture dish), Cd fluxecording was repeated as described above.

d flux of vascular bundles in the leaf petioleThe roots of intact HE and NHE seedlings were preincubated

ith 10 �M CdCl2 for 1 h. The basal leaf was then cut off from theetiole with a sharp blade. After that, the roots and the cut of the leafetiole were incubated in the CdCl2 solution (10 �M) and measur-

ng solution (50 �M CaCl2, pH 5.5) respectively (in different cultureish). The Cd flux was continuously measured for 30 min near theascular bundles of the petiole after 1 h of incubation.

ata analysis

The Cd fluxes were calculated using MageFlux, which was devel-ped by the Xu-Yue company (http://xuyue.net/mageflux).

esults and discussion

In this study, the spatial characteristics and real-time kineticsf Cd transport in the HE and NHE of S. alfredii were demonstratedsing a Cd-selective microelectrode. The results show that regions

and B of the roots in HE S. alfredii exhibited higher Cd influx rateshan those of the NHE after 2 h of 10 �M CdCl2 treatment (Fig. 1).owever, no significant difference in the Cd influx in region C of

he two ecotypes was observed. These results are consistent withhe previous findings, wherein a significantly higher Cd influx wasbserved in the apical region of the roots than at locations furtherack from the apex (Pineros et al., 1998). In addition, the additionf 10 �M CdCl2 to the measuring solution caused a sudden influxf Cd in both ecotypes (Fig. 2a). The peak and mean rates of Cd

nflux in HE were 1.4- and 2.2-fold higher than that of the NHEFig. 2b). Thus, a higher Cd uptake occurs at the onset of Cd expo-ure in the roots of HE S. alfredii. It should be kept in mind that forigh exposure concentrations, short term Cd uptake by the roots is

significant differences between the two ecotypes at P < 0.05.

dominated by cell wall sorption (Redjala et al., 2009, 2010). How-ever, in S. alfredii, a stronger cell wall sorption of Cd was observedin the NHE root system (Lu, 2008). Thus, the higher influx of Cdobserved in the roots of HE is possibly mediated by a more abundantCd-permeable channels and/or transporters in the PM (Lu et al.,2008, 2009). During the 90 min radiotracer experiment, no signifi-cant difference in the unidirectional influx rate of Cd was observedin the roots of the two ecotypes (Lu et al., 2008). Thus, SIET can bean ideal tool for measuring the real-time kinetics of Cd transportbetween accumulators and non-accumulators. However, previousstudies found no significant difference between the Cd flux in theroots of the Cd/Zn hyperaccumulator N. (Thlaspi) caerulescens andthe nonaccumulator Thlaspi arvense using the same Cd-selectivemicroelectrode (Pineros et al., 1998). In addition, Cd influx was notobserved until the roots were exposed overnight to Cd treatmentin the two Thlaspi species (Pineros et al., 1998). This distinctionbetween S. alfredii and N. (Thlaspi) caerulescens indicates an entirelydifferent temporal characteristic of Cd uptake by the roots in thetwo hyper-accumulators.

Notably, the growing root hair cells (100–150 �m, usuallyexhibited vigorous Cd flux than mature root hairs) of HE S. alfredii,showed an enhanced influx of Cd after 2 h of incubation in 10 �MCdCl2 compared with the root hairs of NHE (Fig. 1b). Root hairsare the main uptake sites of a large number of mineral nutrientsin the roots (Jungk, 2001). Thus, the abundant channels and/ortransporters in the PM of root hair cells can facilitate the uptakeof Cd. A recent study found that root hairs can substantially con-tribute to Cd uptake in barley (Zheng et al., 2011). In addition, hairy

roots can contribute to the hyper-accumulation of Cd in N. (Thlaspi)caerulescens (Nedelkoska and Doran, 2000). Thus, the higher root

3 t Physiology 170 (2013) 355– 359

ha

t2atbretisrus2ss(if

a

Fig. 3. Cd fluxes of leaf petiole vascular bundles in HE and NHE S. alfredii. The Cdfluxes near the vascular bundles in the leaf petiole were continuously measured for30 min before and after pre-exposure of root system to 10 �M CdCl2 for 2 h. Each

Fmfl

58 J. Sun et al. / Journal of Plan

air-mediated Cd influx may be a key contributor to the hyper-ccumulation of Cd in HE S. alfredii.

A rapid root-to-shoot translocation of Cd may also be an impor-ant characteristic of Cd hyperaccumulators (Verbruggen et al.,009). In this study, the root-to-shoot translocation rates of Cd in HEnd NHE S. alfredii were compared using a Cd-selective microelec-rode. The results show a significant efflux of Cd near the vascularundles in the leaf petiole of the two ecotypes of S. alfredii after theoot system was pre-exposed to 10 �M CdCl2 for 2 h (Fig. 3). How-ver, the magnitude of Cd efflux in HE was several folds higher thanhat in NHE (Fig. 3). By contrast, this Cd efflux was not observedn both ecotypes without root system treatment with Cd (Fig. 3),howing that the efflux was caused by Cd translocation from theoots. Our results are consistent with the previous findings obtainedsing the radioactive method, in which HE S. alfredii exhibited aignificantly higher Cd root-to-shoot translocation rate (Lu et al.,008). However, the difference between the 109Cd contents in thehoots of the HE and NHE S. alfredii was not observed until the rootystem was exposed to the 10 �M 109Cd solution for more than 4 hLu et al., 2008; Lu, 2008). Thus, our experimental system may be an

deal tool for monitoring the real-time kinetics of Cd translocationrom the roots to the shoots in other plant species.

The absorbed Cd in the shoots should be handled more appropri-tely to prevent toxicity (Verbruggen et al., 2009). Previous studies

ig. 4. Transient Cd kinetics of mesophyll tissues (a), isolated mesophyll protoplasts (b)ean of five independent experiments; the bars represent the standard error of the mea

uxes in a–c, respectively. Columns labeled with a and b indicate significant difference be

point represents the mean of four individual plants; the bars represent the standarderror of the mean. The inset images show the measuring position.

suggested that Cd in HE S. alfredii plants accumulate primarily in theparenchyma tissues, including the pith, cortex, and mesophyll. In

addition, the vacuoles of mesophyll cells may serve as the primarystorage sites for Cd (Tian et al., 2011). In this study, the kineticsof Cd uptake in mesophyll tissues, isolated mesophyll protoplasts,

and intact vacuoles (c) in response to CdCl2 treatment. Each point represents then. The inset images show the measuring position. (d–f) Peak and mean rates of Cdtween the two ecotypes at P < 0.05.

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J. Sun et al. / Journal of Plan

nd intact vacuoles from HE and NHE S. alfredii were compared.ig. 4a shows a markedly enhanced influx of Cd in the mesophyllissues of both ecotypes. On the other hand, the peak and meanates of Cd influx in HE were significantly higher than those in NHEFig. 4d). To eliminate the effect of the cell wall on Cd transport,rotoplasts were isolated from mesophyll tissues to measure thed flux directly across the PM. A steady influx of Cd (mean rate,.2 pmol cm−2 s−1) was detected in the HE protoplasts after expo-ure to 10 �M CdCl2 (Fig. 4b and e). This result is similar to thatbserved in the mesophyll tissues. However, the Cd flux in theHE protoplasts exhibited a two-phase response, with an instan-

aneous increase in influx followed by a continuous drift towardfflux (Fig. 4b and e). These results show that the strong Cd uptaken the HE mesophyll cells is mediated by the Cd-permeable chan-els and/or transporters in the PM. By contrast, the observed Cd

nflux in the NHE mesophyll tissues may be attributed to cell walld sorption. A significantly enhanced influx of Cd at a mean ratef 3.0 pmol cm−2 s−1 was observed in the intact vacuoles from HEesophyll protoplasts (Fig. 4c and f). This phenomenon was not

bserved in the NHE vacuoles. This result clearly indicates that theesophyll cells of HE S. alfredii have a more efficient Cd-transport

ystem in the tonoplast, which transfers the absorbed Cd into theacuoles. Our results are consistent with the high Cd concentrationetected in the vacuoles of mesophyll cells in HE S. alfredii usinghe fluorescence imaging technique (Tian et al., 2011). Although theifferences of Cd transport between the two ecotypes of S. alfrediiere evident, the physiological and molecular mechanisms behind

he faster influx of Cd across the PM and tonoplast of mesophyllells in HE S. alfredii remain largely unknown. Thus, these issuesay be worthwhile topics to pursue in the future using a com-

ination of the Cd-selective microelectrode, fluorescence imaging,nd comparative genomic or proteomic analysis (Lee et al., 2010;eitenmaier and Kupper, 2011).

In summary, innate differences exist between the sorption andransport characteristics of the HE and NHE of S. alfredii. The hyperccumulation ability of HE S. alfredii appears to be regulated by theapid Cd uptake in the specific regions of the roots, including thepical region and root hair cells, faster root-to-shoot translocation,nd a highly efficient PM and tonoplast Cd transport system in theesophyll cells. To the best of our knowledge, this work is the first

o use a Cd-selective microelectrode to study the Cd transport inoot hair cells, mesophyll protoplasts, and intact vacuoles, and islso the first to investigate the translocation of Cd from the rootso the shoots. Our protocol provides an excellent method with aigh degree of spatial resolution for the study of Cd transport athe tissue, cellular, and sub-cellular levels in plants.

cknowledgments

This work was financially supported jointly by the Central Pub-ic Research Institutes Basic Funds for Research and DevelopmentAgro-Environment Protection Institute, Ministry of Agriculture),

he National Science Foundation of China (Grant No. 31200470),he Scientific Research Support Project for Teachers with Doctor’segrees (Jiangsu Normal University, China, No. 11XLR23), the Pri-rity Academic Program Development of Jiangsu Higher Education

iology 170 (2013) 355– 359 359

Institutions, and the Special Fund for Agro-scientific Research inthe Public Interest (201203045).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jplph.2012.10.014.

References

Cosio C, Martinoia E, Keller C. Hyperaccumulation of cadmium and zinc in Thlaspicaerulescens and Arabidopsis halleri at the leaf cellular level. Plant Physiol2004;134:716–25.

He J, Qin J, Long Y, Ma Y, Li H, Li K, et al. Net cadmium flux and accumulation revealtissue-specific oxidative stress and detoxification in Populus canescens. PhysiolPlant 2011;145:50–63.

Jungk A. Root hairs and acquisition of plant nutrients from soil. J Plant Nut Soil Sci2001;164:121–9.

Lee K, Bae DW, Kim SH, Han HJ, Liu X, Park HC, et al. Comparative proteomic analysisof the short-term responses of rice roots and leaves to cadmium. J Plant Physiol2010;167:161–8.

Leitenmaier B, Kupper H. Cadmium uptake and sequestration kinetics in individualleaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens. PlantCell Environ 2011;34:208–19.

Li L, Liu X, Peijnenburg W, Zhao J, Chen X, Yu J, et al. Pathways of cadmium fluxes inthe root of the halophyte Suaeda salsa. Ecotoxicol Environ Saf 2012;75:1–7.

Lu L, Tian S, Yang X, Li T, He Z. Cadmium uptake and xylem loading are activeprocesses in the hyperaccumulator Sedum alfredii. J Plant Physiol 2009;166:579–87.

Lu L, Tian S, Yang X, Wang X, Brown P, Li T, et al. Enhanced root-to-shoot transloca-tion of cadmium in the hyperaccumulating ecotype of Sedum alfredii. J Exp Bot2008;59:3203–13.

Lu L, Tian S, Zhang M, Zhang J, Yang X, Jiang H. The role of Ca pathway in Cduptake and translocation by the hyperaccumulator Sedum alfredii. J HazardMater 2010;183:22–8.

Lu L. Cadmium uptake and translocation in the hyperaccumulator Sedum alfredii H.Ph D dissertation, Zhejiang University, Hangzhou, China; 2008 [in Chinese].

Ma W, Xu W, Xu H, Chen Y, He Z, Ma M. Nitric oxide modulates cadmium influxduring cadmium-induced programmed cell death in tobacco BY-2 cells. Planta2010;232:325–35.

Mohanty M, Dhal NK, Patra P, Das B, Reddy PS. Phytoremediation: a novel approachfor utilization of ironore wastes. Rev Environ Contam Toxicol 2010;206:29–47.

Nedelkoska TM, Doran PM. Hyperaccumulation of cadmium by hairy roots of Thlaspieaeruleseens. Biotechnol Bioeng 2000;67:607–15.

Pilon-Smits E. Phytoremediation. Annu Rev Plant Biol 2005;56:15–39.Pineros MA, Shaff JE, Kochian LV. Development, characterization, and application of

a cadmium-selective micro-electrode for the measurement of cadmium fluxesin roots of Thlaspi species and wheat. Plant Physiol 1998;116:1393–401.

Redjala T, Sterckeman T, Morel JL. Cadmium uptake by roots: contribution ofapoplast and of high- and low-affinity membrane transport systems. EnvironExp Bot 2009;67:235–42.

Redjala T, Sterckeman T, Morel JL. Determination of the different components ofcadmium short-term uptake by roots. J Plant Nutr Soil Sci 2010;173:935–45.

Sun J, Chen S, Dai S, Wang R, Li N, Shen X, et al. NaCl-induced alternations of cellularand tissue ion fluxes in roots of salt-resistant and salt-sensitive poplar species.Plant Physiol 2009;149:1141–53.

Sun J, Wang M, Ding M, Deng S, Liu M, Lu C, et al. H2O2 and cytosolic Ca2+ signalstriggered by the PM H+-coupled transport system mediate K+/Na+ homeostasisin NaCl-stressed Populus euphratica cells. Plant Cell Environ 2010;33:943–58.

Tian S, Lu L, Labavitch J, Yang X, He Z, Hu H, et al. Celllular sequestration ofcadmium in the hyperaccumulator plant species Sedum alfredii. Plant Physiol2011;157:1914–25.

Verbruggen N, Hermans C, Schat H. Molecular mechanisms of metal hyperaccumu-lation in plants. New Phytol 2009;181:759–76.

Yang X, Long X, Ye H, He Z, Calvert DV, Stoffella PJ. Cadmium tolerance and hyperac-cumulation in a new Zn hyperaccumulating plant species (Sedum alfredii Hance).Plant Soil 2004;259:181–9.

Zheng R, Li H, Jiang R, Romheld V, Zhang F, Zhao F. The role of root hairs in cadmiumacquisition by barley. Environ Pollut 2011;159:408–15.