Poplar calcineurin B-like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through...

Original Article

Poplar calcineurin B-like proteins PtCBL10A and PtCBL10Bregulate shoot salt tolerance through interaction withPtSOS2 in the vacuolar membrane

Ren-Jie Tang, Yang Yang, Lei Yang, Hua Liu, Cui-Ting Wang, Meng-Meng Yu, Xiao-Shu Gao & Hong-Xia Zhang

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China

ABSTRACT

The calcineurin B-like protein (CBL) family represents aunique group of calcium sensors in plants. In Arabidopsis,CBL10 functions as a shoot-specific regulator in salt toler-ance. We have identified two CBL10 homologs, PtCBL10Aand PtCBL10B, from the poplar (Populus trichocarpa)genome. While PtCBL10A was ubiquitously expressed atlow levels, PtCBL10B was preferentially expressed inthe green-aerial tissues of poplar. Both PtCBL10A andPtCBL10B were targeted to the tonoplast and expression ofeither one in the Arabidopsis cbl10 mutant could rescue itsshoot salt-sensitive phenotype. Like PtSOS3, both PtCBL10sphysically interacted with the salt-tolerance componentPtSOS2. But in contrast to the SOS3-SOS2 complex at theplasma membrane, the PtCBL10-SOS2 interaction was pri-marily associated with vacuolar compartments. Furthermore,overexpression of either PtCBL10A or PtCBL10B con-ferred salt tolerance on transgenic poplar plants by maintain-ing ion homeostasis in shoot tissues under salinity stress.These results not only suggest a crucial role of PtCBL10s inshoot responses to salt toxicity in poplar, but also provide amolecular basis for genetic engineering of salt-tolerant treespecies.

Key-words: PtCBL10; salt tolerance; transgenic poplar.

INTRODUCTION

Soil salinity is a worldwide environmental constraint thatseverely affects plant growth in general and agricultureproductivity in particular. Usually, sodium chloride (NaCl)represents the most soluble and abundant salt in saline soilsencountered by plants (Munns & Tester 2008). Most plantspecies including trees are sensitive to high concentrations ofsodium (Na+) that cause water deficiency imposed by osmoticstress and cellular damages inflicted by ionic toxicity(Hasegawa et al. 2000). In order to cope with the varyingexternal Na+ concentrations, higher plants are equipped witha variety of transport proteins that mediate Na+ absorp-tion, extrusion, translocation and sequestration (Horie &Schroeder 2004; Apse & Blumwald 2007). Na+ entry into

plant cells is supposed to be mediated by non-selective cationchannels (NSCCs), although their molecular identities havenot been unravelled yet (Demidchik & Maathuis 2007).Whenthe cytoplasmic Na+ concentration reaches a toxic level, theexcessive Na+ can potentially be extruded by Na+/H+

antiporters located in the plasma membrane (Qiu et al. 2003),or sequestered into vacuoles by tonoplast Na+/H+ antiporters(Blumwald & Poole 1985).These active Na+-transporting pro-cesses contribute to Na+ detoxification in plant cells (Zhu2003).At the whole-plant level,Na+ tends to be translocated tovarious plant tissues and properly redistributed within theplant. It is believed that vascular long-distance Na+ transportalso plays a critical role in plant salt tolerance (Munns &Tester 2008). Recent studies have shown that HKT-type Na+

influx transporters function in mediating the retrieval of Na+

from the xylem, thus limiting Na+ transport from root to shootand protecting leaf tissues from salinity stress (Ren et al. 2005;Møller et al. 2009; Munns et al. 2012).

In addition to the ion transporting system that directlyhandles Na+ distribution, plants have evolved a globalnetwork of signal transduction pathways, by which theyrespond and adapt to environmental salt stress. The calcium(Ca2+)-dependent salt overly sensitive (SOS) pathway hasemerged as a central player in the regulation of ion homeo-stasis under saline conditions. It has been suggested that saltstress would elicit a transit increase in cytosolic Ca2+ concen-tration (Knight, Trewavas & Knight 1997), which can be per-ceived by calcium-binding proteins such as SOS3 (Liu & Zhu1998). Upon perception of the specific calcium signal, SOS3regulates the downstream protein kinase SOS2 by increasingits activity and recruiting it to the plasma membrane. TheSOS3-SOS2 complex further activates SOS1, a Na+/H+

exchanger in the plasma membrane, so as to transport Na+

out of the plant cell and prevent its toxic accumulation (Guoet al. 2001; Qiu et al. 2002; Quintero et al. 2002). InArabidopsis, the calcium sensor SOS3 belongs to a group ofcalcineurin B-like proteins (CBLs)/SOS3-like calcium-binding proteins (SCaBPs) that specifically interact with afamily of serine/threonine kinases designated as CBL-interacting kinases (CIPKs)/SOS2-like proteins kinases(PKS; Luan et al. 2002; Gong et al. 2004). While SOS3/CBL4mainly functions in root tissues, CBL10/SCaBP8 appears tobe a shoot-specific regulator of SOS2/CIPK24 in ArabidopsisCorrespondence: H-X. Zhang. E-mail: [email protected]

Plant, Cell and Environment (2014) 37, 573–588 doi: 10.1111/pce.12178

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© 2013 John Wiley & Sons Ltd 573

salt stress tolerance (Kim et al. 2007; Quan et al. 2007). OtherCBL proteins,often coupled with their interacting CIPKs,alsoserve as pivotal calcium sensors in diverse environmentalresponses.Under low potassium (K+) supply conditions,CBL1and CBL9 specifically interact with and activate CIPK23 tostimulate the activity of the shaker-like K+ channel AKT1 (Liet al. 2006;Xu et al. 2006).On the other hand,CBL1/9-CIPK23complex participates in nitrate uptake and sensing processesby phosphorylating the nitrate transporter CHL1/NRT1.1(Ho et al. 2009). Moreover, it has recently been suggested thatthe channel activity and plasma membrane targeting ofAKT2is modulated by the CBL4-CIPK6 complex (Held et al. 2011).The tonoplast-localized CBL2 and CBL3 appear to play criti-cal roles in plant development and intracellular ion homeo-stasis through regulating vacuolar H+-ATPase (V-ATPase)activity (Tang et al. 2012). In addition, overexpression ofCBL5 was shown to improve salt and drought tolerance intransgenic Arabidopsis plants (Cheong et al. 2010).

Although substantial research progress has been made inArabidopsis CBL-CIPK signalling network, very limitedinformation is known with regard to these calcium signallingmodules in other plant species. With the completion ofgenome sequencing and availability of developing genomictools, the genus Populus has become an ideal model plant forforest trees (Tuskan et al. 2006; Jansson & Douglas 2007).Previously, we identified and functionally characterized thegenes encoding the components of SOS pathway (PtSOS1-3)in poplars (Tang et al. 2010). The Populus genome encodesseveral additional PtSOS3-like CBL-type calcium sensorsthat potentially have various functions in response to abioticstresses. In this study, we further analyse the role of PtCBL10sin salt stress adaptation. Based on protein sequence similarityas well as complementation tests in an Arabidopsis mutant,weconclude that PtCBL10A and PtCBL10B are the functionalhomologs of AtCBL10 that mediate shoot salt tolerancein poplar. We also find that, although both PtSOS3 andPtCBL10s physically interact with the protein kinase PtSOS2,they alternatively target PtSOS2 to different subcellular mem-branes. Moreover, we show that overexpression of PtCBL10sand PtSOS3 differentially regulates Na+/K+ homeostasis intransgenic poplar plants to support their survival under salin-ity stress. Our results suggest that the calcium sensorsPtCBL10s and PtSOS3 may exert distinct functions in saltstress adaptation of woody plants.

MATERIALS AND METHODS

Plant materials and growth conditions

The sequenced Populus trichocarpa genotype Nisqually-1and two hybrid aspen clones Yinzhong Yang (Populusalba × Populus berolinensis) and Shanxin Yang (Populusdavidiana × Populus bolleana) were used in this study.Basically, all the poplar materials were amplified bymicropropagation and kept under cool white fluorescent light(40 μmol m−2 s−1) with a 12 h light/12 h dark photoperiodat 21–24 °C during the daytime and 15–18 °C at night.The plantlets were grown in glass containers and monthly

subcultured by aseptically transferring shoot apices onto freshMurashige & Skoog (MS) medium (Murashige & Skoog 1962)supplemented with 0.05 mg L−1 1-naphthaleneacetic acid(NAA) for rooting. One-month-old in vitro grown plantletswere individually transferred into pots filled with a mixture ofsoil, perlite and vermiculite (4:1:1, v/v), and kept in the green-house under a 14 h photoperiod comprising natural daylightsupplemented with lamps (120–150 μEm−2 s−1).The plants werewell irrigated according to the evaporation demand and ferti-lized with one-eighth strength of MS nutrient solutions weekly.

Arabidopsis thaliana and Nicotiana benthamiana plantswere grown in the greenhouse under a 12 h light/12 h darkcycle at 22–23 °C. Sterilized seeds were plated on MS mediumsolidified with 0.8% agar and 7-day-old seedlings were trans-ferred to soil.The cbl10 mutant used in this study is theT-DNAinsertion line SALK_056042 in Col-0 background.

Cloning and gene expressionanalyses PtCBL10s

To isolate the candidate gene sequences encoding CBL10from P. trichocarpa, a blast search was conducted in theupdate poplar genome database (http://www.phytozome.net/poplar) based on the Arabidopsis CBL10 protein sequence(At4G33000.2) obtained from TAIR (http://www.arabidopsis.org/). Two fragments containing PtCBL10A or PtCBL10Bopen reading frame (ORF) were amplified by polymerasechain reaction (PCR) using salt-treated leaf cDNA sample astemplate,and cloned into the EcoRV site in pBlueScript II KS(pKS; Stratagene, La Jolla, CA, USA).After sequence confir-mation and manual revision of the coding sequence (CDS),amino acid sequence alignment was performed using theCLUSTALX program (http://www.clustal.org/) and thetransmembrane domains (TMDs) were predicted byTMHMM (http://www.cbs.dtu.dk/services/TMHMM/) andSOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/). To remove theTMD from PtCBL10A and PtCBL10B, inverse PCR-basedmutagenesis was carried out on the double-stranded pKS-PtCBL10A and pKS-PtCBL10B plasmids, resulting inPtCBL10AΔTMD and PtCBL10BΔTMD, respectively.

For gene expression analysis, total RNA was extractedwith the RNAiso Reagent (Takara, Kyoto, Japan) from rootsand shoots of 1-month-old plantlets, or from various tissues(roots, mature leaves, xylem, phloem and apical buds) of6-month-old poplar grown in the greenhouse. For salt stresstreatment, 3-week-old micropropagated plantlets were sub-jected to the treatment with 200 mm NaCl in the tissue-culture containers, and RNA was isolated at the indicatedtime points. Two micrograms of total RNA was treated withDNase I (Promega, Madison, WI, USA), and then subjectedto reverse transcription reaction using M-MLV reversetranscriptase (TOYOBO, Shang, China) at 42 °C for 1 h. Theresultant cDNA was used for PCR amplification with thegene-specific primers (Supporting Information Table S1).

Quantitative real-time PCR (qRT-PCR) analysis wascarried out with the Rotor-Gene 3000 system (CorbettResearch, Sydney, Australia). Each PCR mixture contained10 μL SYBR-Green PCR Master Mix (TOYOBO), 0.5 μm

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© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 573–588

gene-specific primers and 50–100 ng cDNA sample in a finalvolume of 20 μL. Data analysis was performed with Rotor-Gene software version 6.0. The relative value of expressionlevel was calculated based on the comparative thresholdcycle method using the poplar EF1β as an internal control,and was further normalized to the control expression valuesmeasured in the roots or at 0 h salt treatment. All the PCRprimers are listed in Supporting Information Table S1.

Subcellular localization studies

To determine the subcellular localization of PtCBL10s, thecoding region of PtCBL10A, PtCBL10B, PtCBL10AΔTMD,PtCBL10BΔTMD or PtSOS3 was in-frame fused to theyellow fluorescent protein (YFP) sequence via the SmaI-SalIsite in the pA7-YFP plasmid and transferred into poplarmesophyll protoplasts by polyethylene glycol (PEG)-mediated transfection (Yoo, Cho & Sheen 2007). YFP fluo-rescence was imaged after the protoplasts were incubated at23 °C for 16 h by the LSM510 META confocal laser scanningmicroscope (Carl Zeiss, Jena, Germany). The filter settingsare as follows: YFP, Ex 514 nm Em–1 BP 535–600 nm; chloro-phyll, Ex 488 nm Em–1 LP 650 nm.

Yeast two-hybrid and Ras recruitment assays

The CDSs of PtSOS2, PtSOS3, PtCBL10A, PtCBL10B andrelative mutant variants were cloned into the DNA-bindingdomain (BD) vector pGBKT7 and the activation domain(AD) vector pGADT7, respectively. PtSOS2 lacking theNAF/FISL motif (PtSOS2ΔF) was generated as describedpreviously (Tang et al. 2010). Various plasmid combinationswere transformed into yeast strain AH109 and selected onSD agar medium devoid of leucine and tryptophan (SD/-Leu/-Trp). Tenfold serial dilutions of the yeast cells wereplated onto selection medium for interaction assays (SD/-Leu/-Trp/-His and SD/- Leu/-Trp/-His/-Ade).

For the yeast Ras recruitment system (RRS) assay, theCDSs of PtCBL10A, PtCBL10B, PtSOS3, PtCBL1, AtSOS3and AtCBL10 were cloned into the p425GPD vector, respec-tively (Mumberg, Muller & Funk 1995). RAS-PtSOS2 andRAS-PtSOS2ΔF were constructed as described previously(Tang et al. 2010). Different combinations of the generatedconstructs were transferred into the yeast strain cdc25-2(MATα, ade2, his3, leu2, lys2, trp1, ura3, cdc25-2) and selectedon SD/–Ura/-Leu medium. For interaction analysis, serialdecimal dilutions of yeast cultures were spotted onto yeastextract peptone dextrose (YPD, 1% yeast extract, 2%peptone, 2% dextrose) plates at permissive (24 °C) andrestrictive (36 °C) temperatures, respectively. At least sixindependent yeast colonies were selected in this assay.

Bimolecular fluorescence complementation(BiFC) assay

A pair of improved BiFC constructs were generated anddesignated as p13SP-nYNT and p23SP-cYCT (Supporting

Information Fig. S1). The cDNA of PtSOS2 and PtSOS2ΔFwas cloned into p13SP-nYNT via the BamHI-SalI site, whilethe coding region of PtCBL10A, PtCBL10B, PtSOS3,PtCBL10AΔTMD and PtCBL10BΔTMD was cloned intop23SP-cYCT via the SmaI-SpeI site, respectively. Forplant transient expression, different combinations ofAgrobacterium strains carrying BiFC constructs wereco-infiltrated with p19 strain into N. benthamiana leaves(Waadt et al. 2008). Three days after infiltration, protoplastswere isolated with the enzyme solution containing 0.4 m man-nitol, 20 mm MES-K, 10 mm CaCl2, 5 mm β-mercaptoethanol,0.1% bovine serum albumin, 1% cellulase R10, 0.3%macerozyme R10,pH 5.7.YFP fluorescence in the leaf epider-mal cells as well as the protoplasts was imaged with a confocallaser scanning microscope (LSM510; Carl Zeiss).

Arabidopsis transformation andcomplementation test

To generate plant transformation constructs, theCDS of PtCBL10A, PtCBL10B, PtCBL10AΔTMD,PtCBL10BΔTMD and PtSOS3 was cloned into a modifiedpCAMBIA-1301 vector via the BamHI-SalI site, respectively(Supporting Information Fig. S2). The resultant plasmidswere introduced into the Agrobacterium tumefaciens GV3101strain for Arabidopsis transformation in the cbl10 mutantbackground by the floral dipping method (Clough & Bent1998). Putative transgenic plants were screened on MSmedium supplemented with 30 μg L−1 hygromycin and thentransferred to soil for propagation. Hygromycin-resistantplants of T2 generation were subjected to transgene expres-sion analyses. About 10–20 T2 transgenic lines for eachtransgene were subjected to preliminary complementationtests. Selected homozygous T3 transgenic plants, together withwild-type (WT) Col-0 and the cbl10 mutant,were further usedfor salt sensitivity assays on MS agar plates supplemented with60 or 120 mm NaCl.

Poplar transformation and transgeneconformation

The hybrid poplar clone Shanxin (P. davidiana × P. bolleana)was transformed as described previously (Wang et al. 2011)with minor modifications. Briefly, leaf explants excisedfrom 1-month-old plantlets were inoculated in theAgrobacterium (EHA105 strain) culture for 10 min andthen plated on solid MS medium supplemented with0.4 mg L−1 6-benzylaminopurine (6-BA),0.1 mg L−1 NAA and100 μm acetone-syringone. After 2 d of co-cultivation withAgrobacterium, the explants were biweekly transferred ontoMS medium containing 0.4 mg L−1 6-BA, 0.1 mg L−1 NAA,0.01 mg L−1 thidiazuron (TDZ), 400 mg L−1 Timentin and10 mg L−1 hygromycin for selective regeneration. When theregenerated shoots reached 1 cm tall, they were separatedfrom the calli and transferred onto the rooting medium(MS supplemented with 0.05 mg L−1 NAA and 5 mg L−1

hygromycin).The rooted plantlets were transplanted into soilwhen they were over 5 cm in height.

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The presence of the transgene in different transgenic lineswas first assessed by PCR amplification using primers specificfor 35S promoter and PtCBL10 cDNA (Supporting Informa-tion Table S1). To verify the expression of transgene inthe PCR-positive plants, we detected the activity of β-glucuronidase (GUS) reporter gene that was co-transformedby histochemical staining. Leaves were detached from poplarplants and incubated at 37 °C for 12 h in GUS staining buffer(50 mm sodium phosphate, pH 7.0, 0.5 mm ferricyanide,0.5 mm ferrocyanide, 0.1% Triton X-100, 1 mm 5-bromo-4-chloro-3-indolyl-β-D-glucuronide). The stained materialswere incubated in 75% ethanol overnight to remove thechlorophyll and then kept in 95% ethanol (Jefferson,Kavanagh & Bevan 1987).

Salt stress tolerance tests of transgenicpoplar plants

WT and transgenic plants with the same micropropagatedgeneration were transferred to soil and grown in the green-house. After 6 weeks, WT plants and transgenic lines withsimilar size and growth status were chosen and subjected tosalt stress treatment for 1 month. Every 3 d, the control groupwas watered with a diluted nutrient solution containingone-eighth strength of MS salts while the treated group waswatered with the same solution supplemented with NaCl.Theconcentrations of NaCl supplementation were stepwiseincreased by 25 mm every 3 d until it reached 100 mm. Afterbeing treated with 100 mm NaCl for 21 d, the plants wereirrigated with fresh water and recovered for 3 d, and thenphotographed. Plant growth was determined by measuringthe plant height from the top of the shoot apex to the base of

the stem, as well as the fresh weight of the above-ground partof the poplar plants.

Determination of Na+ and K+ contents

Ion contents were measured in WT and transgenic plants atthe end of salt stress treatment. Plant materials were har-vested and pooled in roots, stems and leaves. The sampleswere dried for 48 h at 80 °C, milled to fine powder, weighedand digested with concentrated HNO3. Na+ and K+ concen-trations were determined in the digested liquid using anatomic absorption spectrophotometer (Hitachi Z-8000;Tokyo, Japan).

RESULTS

PtCBL10A and PtCBL10B encode two putativecalcium sensors in Populus

A BLAST search against AtCBL10 protein sequence re-sulted in the identification of two homologs desig-nated as PtCBL10A (Potri.003G035400.1) and PtCBL10B(Potri.006G230200.1) in the Populus genome. The proteinsequence of PtCBL10A and PtCBL10B shares 67% sequenceidentity, and displays 71 and 67% identity with that ofAtCBL10, respectively. Similar to AtCBL10, both PtCBL10Aand PtCBL10B are characterized by four conserved EF-handmotifs that are supposed to be responsible for Ca2+ binding.However, they are distinct from other CBL-type calciumsensors such as PtSOS3 (Potri.015G013100.2), in that theyharbour an extended N-terminal peptide that potentiallycontains a TMD (Fig. 1). As a common feature of all theseCBLs, PtCBL10s have the conserved single serine residue

Figure 1. Sequence alignment of Populus and Arabidopsis CBL10 and SOS3 proteins. The deduced amino acid sequences of PtCBL10A,PtCBL10B, AtCBL10, PtSOS3 and AtSOS3 are compared. The sequences were aligned using CLUSTALX software. The putativetransmembrane domain of CBL10 is framed and four EF-hand motifs are underlined. The lipid modification residues of SOS3 are labelled bythe asterisks. The triangle indicates the conserved serine phosphorylation site.



in the C-terminus (Fig. 1), which could potentially bephosphorylated by CIPK-type kinases (Du et al. 2011;Hashimoto et al. 2012). The fact that Arabidopsis genomeencodes only one copy of CBL10 implies that Populus mighthave duplicated this gene during the long evolution, whichenables its better adaptation to the fluctuating environmentsover a long lifespan.

Expression patterns of PtCBL10A and PtCBL10Bin Populus

As a first step to functionally characterize PtCBL10s, weexamined the transcript abundance of PtCBL10 genes invarious poplar tissues by RT-PCR analysis. In P. trichocarpa,PtCBL10A exhibited relatively low and uniform expressionlevels in all tested tissues (Fig. 2a). While the expression ofPtCBL10B was only marginally detectable in the root, a verystrong expression was observed in the aerial tissues of thepoplar including lamina, petiole, xylem, phloem and apex(Fig. 2a). Oppositely, PtSOS3 was preferentially expressedin root tissues, which is consistent with the earlier report(Tang et al. 2010). Similar expression pattern in P. alba × P.berolinensis corroborated the spatial expression patterns ofthese three calcium sensor genes in poplar species (Fig. 2b).We further confirmed the expression pattern of PtCBL10Aand PtCBL10B, as well as that of PtSOS3 in poplar by qRT-PCR. Indeed, while the expression of PtCBL10A was com-parable in roots and shoots, PtCBL10B was predominantlyexpressed in shoot tissues (Fig. 2c). Contrary to PtCBL10B,the expression of PtSOS3 in roots was much higher than thatin shoots (Fig. 2c).Thus, considering the low basal expressionlevel of PtCBL10A, the integral expression of PtCBL10should be largely in green tissues, which is complementarywith that of PtSOS3 in root tissue.

To examine whether expression of PtCBL10 in poplar isregulated by salt stress, total RNA was extracted from theplantlets of P. alba × P. berolinensis treated with 200 mmNaCl for increasing periods of time. The transcript level ofPabCBL10A and PabCBL10B was analysed by qRT-PCR.The expression of PabCBL10A was steadily induced by saltfrom 0 to 6 h, and reached a maximal threefold induction at12 h (Fig. 2d). In the meantime, the transcript of PabCBL10Bdid not change significantly during the NaCl treatment(Fig. 2d).

Both PtCBL10A and PtCBL10B are targeted tothe vacuolar membrane

Subcellular localization of a Ca2+ sensor tends to play animportant role in its biological functions. To investigate thelocalization of PtCBL10 proteins in poplar cells, transientexpression of YFP fusion proteins in poplar mesophyllprotoplasts was analysed by confocal microscopy. Asexpected, the empty YFP alone was ubiquitously distributedin the cell (Fig. 3). PtCBL10A-YFP and PtCBL10B-YFPsignals revealed a clear vacuolar membrane localization ofthe fusion proteins, which intracellularly surrounded thechloroplasts (Fig. 3). This tonoplast localization could be

Figure 2. Expression profiles of PtCBL10A and PtCBL10B indifferent tissues of poplar and in response to salt stress. (a–b)Reverse transcription PCR (RT-PCR) analysis of PtCBL10s andPtSOS3 gene expression in (a) Populus trichocarpa and (b)Populus alba × Populus berolinensis. The cDNA samples derivedfrom various tissues were used as templates for polymerase chainreaction (PCR ) with the gene-specific primers at indicatednumbers of cycles. Genomic DNA was used as a control templateto rule out its possible contamination in each cDNA sample. PCRproducts were visualized by ethidium bromide staining after theagarose-gel electrophoresis. The elongation factor gene EF1β wasemployed as an internal control. (c) Quantitative RT-PCR analysesof PtCBL10s and PtSOS3 expression in the root or shoot tissuesof both P. trichocarpa (Pt) and P. alba × P. Berolinensis (Pab).(d) qPCR analyses of PtCBL10s in response to salt stress.Three-week-old micropropagated plants were treated with 200 mmNaCl. Samples were collected at 0, 1, 3, 6, 12 and 24 h after theinitiation of treatment. Total RNA was extracted and RT-PCRanalyses were performed with PtCBL10A and PtCBL10Bgene-specific primers. The relative expression was normalized byEF1β and relative to the control value measured at 0 h. Datarepresent the average of three independent experiments ± standarddeviation (SD, n = 3).



Figure 3. Subcellular localization of PtCBL10A and PtCBL10B. Confocal laser scanning microscopy images of poplar mesophyllprotoplasts transiently expressing either yellow fluorescent protein (YFP) alone or PtCBL10A-YFP, PtCBL10B-YFP, PtSOS3-YFP,PtCBL10AΔTMD-YFP, PtCBL10BΔTMD-YFP fusion proteins under the control of the 35S promoter. The YFP signals and chloroplastauto-fluorescence were labelled by green and red colours, respectively. Overlay of both images was shown in the third column. Images ofbright field from the same cell are shown in the fourth column. Scale bars = 10 μm.



further verified by comparison with the labelling of PtSOS3-YFP, which largely resided in the plasma membrane withsome leaky expression in the nucleus (Fig. 3). Deletion of theputative hydrophobic domain in the N-terminus of eitherPtCBL10A or PtCBL10B led to a diffused fluorescent label-ling in cytoplasm and nucleus, similar to that of YFP alone(Fig. 3). These results indicate that PtCBL10 proteins areprimarily associated with the vacuolar membrane and thislocalization requires the N-terminal TMD for their propertargeting. In addition, we imaged the localization of all theseYFP fusions in the leaf epidermal cells and mesophyllprotoplasts from transiently transformed N. benthamianaleaves. In this system, PtSOS3 was more prominently local-ized in plasma membrane, and PtCBL10s were not only tar-geted to the tonoplast but also present in the endosomalcompartments labelled by dynamic punctate structures(Supporting Information Fig. S3).

Both PtCBL10A and PtCBL10B can functionallycomplement the Arabidopsis cbl10 mutant

The Arabidopsis cbl10/scabp8 mutant displays hypersensitiv-ity to high external Na+ concentrations specifically inshoots (Kim et al. 2007; Quan et al. 2007). To assess whetherPtCBL10s might have similar functions as the Arabidopsisortholog, we heterologously expressed PtCBL10A orPtCBL10B in the cbl10 background. In parallel, PtSOS3 wasintroduced into cbl10 mutant as well. RT-PCR analysis con-firmed the expression of PtCBL10A, PtCBL10B or PtSOS3in the cbl10 mutant, respectively (Fig. 4a). In the absence ofNaCl, seedlings of all the different genotypes exhibiteduniform growth status (Fig. 4b). Under mild salt stress(60 mm NaCl), shoot growth of cbl10 mutant was evidentlyinhibited compared with WT, while expression of PtCBL10Aor PtCBL10B but not PtSOS3 suppressed this shoot saltsensitivity (Fig. 4c). In 120 mm NaCl treatment, PtCBL10Aor PtCBL10B expression could still rescue the more severelyretarded growth of cbl10 (Fig. 4d). A statistical analysis ofshoot fresh weights (Fig. 4e) and primary root lengths(Fig. 4f) showed that salt sensitivity of cbl10 was largelylocated in the shoot tissues, which could be considerablysuppressed by PtCBl10A or PtCBL10B transgene undersalinity conditions. More than 10 independent transgeniccbl10 lines harbouring PtCBL10A or PtCBL10B were testedand showed similar level of salt tolerance to that of WTplants. By contrast, no transgenic lines were found to fullyrescue the salt sensitivity of cbl10 among 20 lines expressingPtSOS3. These results suggest that both PtCBL10A andPtCBL10B are functional orthologs of AtCBL10.

To determine the functional importance of tonoplastlocalization of PtCBL10s in salt tolerance, we also con-structed transgenic lines expressing the truncated versions ofPtCBL10s (PtCBL10AΔTMD and PtCBL10BΔTMD) thathad already been shown to fail to reside in the vacuolarmembrane (Fig. 3). Seeds of T2 generation from 20 independ-ent T1 transgenic lines in the cbl10 background were sub-jected to the complementation tests.When the seedlings weretreated with 60 or 120 mm NaCl, none of them could rescue

the salt-sensitive phenotype in shoots (Supporting Informa-tion Fig. S4), suggesting that proper targeting of PtCBL10s isrequired for salt stress adaptation.

PtCBL10 interacts with PtSOS2 in thevacuolar membrane

In Arabidopsis, the protein kinase SOS2/CIPK24 interactswith both SOS3/CBL4 and CBL10 to mediate salt tolerance.Here we used yeast two-hybrid assay to test the physicalinteraction between PtSOS2 (Potri.018G130500.3) and itsupstream calcium sensors. While PtSOS3 showed stronginteraction with PtSOS2 in the yeast AH109 strain, the full-length PtCBL10A or PtCBL10B only had weak or little inter-action (Fig. 5a). We reasoned that the hydrophobic regionof PtCBL10s might prevent AD fusions from enteringthe nucleus. After the putative TMD was removed, bothPtCBL10AΔTMD and PtCBL10BΔTMD could stronglyinteract with PtSOS2 in our yeast two-hybrid system(Fig. 5a). However, this interaction was eliminated whenPtSOS2 lost the NAF/FISL motif that is essential for CBL–CIPK interaction (Albrecht et al. 2001; Guo et al. 2001).

Moreover, we employed the yeast RRS (Broder, Katz &Aronheim 1998) to monitor the membrane targeting ofPtSOS2 by CBL-type calcium sensors. Expression of RAS-PtSOS2 fusion alone did not rescue thermo-sensitivity ofthe yeast strain cdc25-2 that is deficient in the Ras guanylnucleotide exchange factor, indicating that RAS-PtSOS2 iscytosolic in the yeast. Co-expression of RAS-PtSOS2 in com-bination with PtSOS3, PtCBL1 or AtSOS3, respectively,restored the thermo-tolerance of the yeast mutant, suggest-ing that PtSOS2 could efficiently be targeted to the plasmamembrane through physical interaction with PtSOS3,PtCBL1 or AtSOS3. However, neither PtCBL10A norPtCBL10B could substantially recruit RAS-PtSOS2 to theplasma membrane as it was shown that yeast strains express-ing RAS-PtSOS2 and PtCBL10A or PtCBL10B did not growwell at the restrictive temperature (Fig. 5b). These resultsimply that unlike PtSOS3-PtSOS2 at the plasma membrane,PtCBL10A and PtCBL10B may form an alternative complexwith PtSOS2 elsewhere.

To further address the spatial specificity of PtCBL10-PtSOS2 complex formation, we imaged the in planta interac-tion between PtCBL10s and PtSOS2 through BiFC analysis.As shown in Fig. 6, the YFP signal resulting from PtSOS2interaction with PtCBL10A or PtCBL10B revealed a clearlocalization of PtCBL10-PtSOS2 complex formation at thevacuolar membrane, which is distinct from the plasma mem-brane pattern displayed by the PtSOS3-PtSOS2 interaction.Removal of the NAF/FISL motif from PtSOS2 failed toproduce obvious signal in the BiFC analyses of PtSOS2ΔFand PtCBL10 interaction. On the other hand, removal of theTMD from PtCBL10A or PtCBL10B did not abolish theinteraction fluorescence but retained it in the cytosol andnucleus (Fig. 6). These results suggested that PtSOS2 inter-acts with PtCBL10 via the NAF/FISL motif, and the putativeTMD of PtCBL10 determines that the interaction takesplace at the vacuolar membrane.



Figure 4. Functional complementation of the Arabidopsis cbl10 mutant by PtCBL10s. (a) Reverse transcription PCR analysis of transgeneexpression or AtCBL10 in wild-type (WT) and cbl10 mutant Arabidopsis plants. The ACTIN2 gene was used as a control. (b–d) Four-day-oldseedlings of WT, cbl10 and cbl10 expressing PtCBL10A, PtCBL10B or PtSOS3 were transferred onto Murashige & Skoog medium containing(b) 0, (c) 60 or (d) 120 mm NaCl. The photographs were taken on the 10th day of treatment. (e) Shoot fresh weight and (f) primary root lengthof all the seedlings were measured at the end of salt treatment. Error bars represent SE from triplicate experiments. Scale bars = 10 mm.



Figure 5. PtCBL10s and PtSOS2 interactions in yeast. (a) Yeast two-hybrid assay of interactions between PtCBL10s and PtSOS2. The yeaststrain AH109 was co-transformed with various combinations of binding domain (BD)- and activation domain (AD)-fusion constructs asindicated. Yeast cells were plated on medium lacking Leu and Trp as a control (left panel). Yeast growth on the selective medium lackingLeu, Trp and His was used to monitor the interaction (middle panel). A more stringent assay was performed on the selective medium lackingLeu, Trp, His and Ade (right panel). Interaction between PtSOS3 and PtSOS2 serves as a positive control. (b) Recruitment of PtSOS2 to theplasma membrane by PtSOS3 but not by PtCBL10s. Plasmid combinations as indicated on the left were expressed in the yeast cdc25-2mutant strain. Three microlitres of serial decimal dilutions were spotted onto yeast extract peptone dextrose solid medium at the permissive(24 °C) or at the restrictive (36 °C) temperature. Yeast cells that are able to grow at 36 °C indicate interaction of the corresponding twoproteins at the plasma membrane.



Figure 6. Bimolecular fluorescence complementation (BiFC) analysis of PtCBL10 and PtSOS2 interaction in planta. Various combinationsof YFPN and YFPC fusion constructs as indicated on the left were co-transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. The images were analysed with a Zeiss confocal microscope. The panels in the first column depict the yellow fluorescentprotein (YFP) signals in intact epidermal cells. The panels in the third column show the YFP fluorescence in protoplasts prepared from thesame leaf. The panels in the second and fourth columns are the respective bright-field images. White arrows point to vacuolar membrane thatinvaginate away from the plasma membrane.



Overexpression of PtCBL10s and PtSOS3enhances salt tolerance in transgenicpoplar plants

In order to approach the function of PtCBL10s in the woodyplant, transgenic poplar plants overexpressing PtCBL10A orPtCBL10B were generated. More than 10 independent trans-formants were successfully obtained for each gene construct(Supporting Information Fig. S2), and the integration of bothtransgenes into the poplar genome was confirmed by PCRanalysis (Supporting Information Fig. S5a). Histochemical

staining for GUS activity verified the stable and constitutiveexpression of the co-transformed gusA gene in transgenicpoplar leaves (Supporting Information Fig. 5b). Furtheranalysis by qRT-PCR quantified the differential expressionlevels of PtCBL10A and PtCBL10B in selected transgenicpoplar plants (Fig. 7c,d). It seemed that PtCBL10A gainedgreater increment in its expression than did PtCBL10B,presumably because of its much lower basal expression inWT poplar. We selected two independent transgenic linesoverexpressing PtCBL10A (LA12, LA24) or PtCBL10B(LB1, LB12) for subsequent phenotypic analysis.

Figure 7. Overexpression of PtCBL10A or PtCBL10B enhances salt tolerance in transgenic poplar plants. (a) Growth of wild-type (WT) aswell as four independent transgenic lines LA12, LA24, LB1 and LB12 in the absence of salt stress for 1 month. (b) Effects of salinity on thegrowth of WT, LA12, LA24, LB1 and LB12. The poplar plants were treated with gradually increased concentrations of NaCl up to a finalconcentration of 100 mm for 21 d. After 3 d of recovery from the salt stress, representative plants were chosen and photographed. Scalebars = 10 cm. (c) Quantitative real-time PCR (qRT-PCR) analysis of PtCBL10A expression in WT and six independent transgenic lines.(d) Quantitative RT-PCR analysis of PtCBL10B expression in WT and five independent transgenic lines. (e) Measurements of the plantheight of WT, LA12, LA24, LB1 and LB12 at the end of 0 or 100 mm NaCl treatment. (f) Quantification of the shoot biomass of WT,LA12, LA24, LB1 and LB12 at the end of 0 or 100 mm NaCl treatment. Values are means ± SD (n = 6) of two independent experiments.Asterisks indicate significant difference in comparison to WT (Student’s t-test, * P < 0.05).



Overexpression of PtCBL10A or PtCBL10B did notchange the overall development or plant morphology of thetransgenic poplar as transgenic lines LA12, LA24, LB1 andLB12 all grew well under normal growth condition (Fig. 7a).However, after the plants were treated with 100 mm NaCl for1 month, obvious differences were observed between WTand transgenic plants. While WT plants were stunted in thegrowth and became wilted, transgenic lines appeared to beless impaired by salt stress (Fig. 7b). Although salt stressconsiderably affected the growth of all the plants in general,transgenic plants had a bigger stature and retained a signifi-cantly greater shoot biomass than the WT plants (Fig. 7e,f).These results demonstrated that overexpression ofPtCBL10s in poplar could enhance salt tolerance.

PtCBL10s and PtSOS3 overexpressiondifferentially regulates tissue Na+ andK+ distribution

We also generated transgenic poplar plants in which PtSOS3was constitutively expressed (Supporting InformationFig. S6). Two independent lines L38 and L314 with very highPtSOS3 expression levels were chosen for salt tolerance test.

Compared with WT poplar, these two transgenic lines dis-played improved salt tolerance, as revealed by the measure-ment of plant height and biomass under salinity (SupportingInformation Fig. S6).

In order to understand how these introduced genesimproved salt-tolerant capacity in transgenic plants, we ana-lysed the distribution of Na+ and K+ contents in differentplant tissues. Under normal growth condition without addi-tional NaCl supplementation, poplar plants seemed todeposit most of Na+ in the root and accumulate more K+ inthe leaves. Both WT and transgenic LA12, LB12 lines con-tained approximately equal Na+ and K+ contents in root, leafand stem tissues (Fig. 8a,b). Surprisingly, L314 exhibited adifferent pattern of Na+ distribution; less Na+ in the root andmore in the leaves and stem. Under saline condition, Na+

content increased in all plant tissues accompanied by adecrease in K+ content. However, Na+ content in LA12 andLB12 was significantly lower than that in WT in leaves butnot in roots whereas L314 accumulated less Na+ only in roots.In the stem, LA12, LB12 and L314 all retained more Na+ thanWT (Fig. 8c). Generally, transgenic and WT plants accumu-lated equivalent levels of K+, except that L314 had a slightlyhigher K+ content in the root during salt stress (Fig. 8d).

Figure 8. Na+ and K+ contents in roots, stems and leaves of wild-type (WT) and transgenic poplar plants. At the end of 0 or 100 mm NaCltreatment, plant materials were harvested and pooled in roots, stems and leaves for the measurements of Na+ and K+ contents. (a) Na+

contents of different tissues at 0 mm NaCl. (b) K+ contents of different tissues at 0 mm NaCl. (c) Na+ contents of different tissues at 100 mmNaCl; (d) K+ contents of different tissues at 100 mm NaCl. Values are means ± SD (n = 4) from two independent experiments. Asterisksindicate statistically significant difference in comparison to WT (Student’s t-test, * P < 0.05, ** P < 0.01).



Taken together, our data suggest that PtCBL10 and PtSOS3favourably regulate Na+/K+ homeostasis under salt stress butfulfil distinct regulatory roles in a tissue-specific manner.

DISCUSSION

Trees are perennial organisms that are likely to encountercontinual environmental stresses during their lifetime.Among various abiotic stresses, salinity is a common limitingfactor to tree growth and productivity. Owing to their sessilenature, trees are not able to move away from high salinity.Instead, they can trigger an array of stress signallingpathways to modulate a cascade of adaptive changes in thetranscriptome, proteome and metabolome, leading to saltstress resistance (Brinker et al. 2010; Janz et al. 2010).

It has been well documented that Ca2+ serves as an impor-tant secondary messenger in plant salt stress signalling(Knight et al. 1997; Kiegle et al. 2000; Kudla, Batistic &Hashimoto 2010; Sun et al. 2010). Cellular calcium signals areperceived and transmitted by a toolkit of calcium-bindingsensor proteins such as calmodulin (CaM) and CaM-like(CML) protein, calcium-dependent protein kinase (CDPK)as well as calcineurin B-like (CBL) protein (Cheng et al. 2002;Luan et al. 2002; McCormack, Tsai & Braam 2005). Amongthem, plant CBL proteins display sequence similarity toneuronal Ca2+ sensors (NCS) and calcineurin B subunit(CNB) from animals and yeast (Kudla et al. 1999). Because allthe CBL proteins contain four highly conserved EF-handdomains with invariable spacing in the arrangement, theirsequence specificity largely relies on their N- and C-terminaldivergence (Kolukisaoglu et al. 2004). Compared with otherCBL family members in Arabidopsis,CBL10 is distinct in thatit harbours a relatively large and partially hydrophobicN-terminal domain. Remarkably, because of the absence inlower land plants such as Physcomitrella and Selaginella,genes coding for CBL10 probably originated from floweringplants (Weinl & Kudla 2009). In this study, we isolated andcharacterized two CBL10 isoforms from the woody plantPopulus. Heterologous expression of either PtCBL10A orPtCBL10B could rescue salt sensitivity of an Arabidopsiscbl10 knockout allele (Fig. 4), demonstrating that they arefunctional homologs of AtCBL10 and are involved in plantsalt stress tolerance. The double copies of CBL10 gene inpoplar may result from evolutionary adaptation of perennialtree plants to the fluctuating and harsh environments overtheir long lifespan. Indeed, the genome of the halophyteThellungiella parvula, a close relative of Arabidopsis, containshigher copy numbers of orthologous genes related to stresstolerance including CBL10, which enables its colonization ofsaline and barren habitats (Dassanayake et al. 2011).

CBL-type calcium sensors can be grouped into two sub-classes according to their N-terminal features that generallydetermine their subcellular localizations. In Arabidopsis,the first subclass consists of AtCBL1, AtCBL4, AtCBL5,AtCBL8 and AtCBL9, most of which harbour motifs for N-myristoylation and S-acylation. Dual lipid modification ofthese CBL proteins anchors them to the plasma membranewhere they fulfil their biological functions (Ishitani et al. 2000;

Batistic et al. 2008). The second subclass is composed ofAtCBL2, AtCBL3, AtCBL6, AtCBL7 and AtCBL10 thatcontain variable N-terminal extensions.With the exception ofAtCBL7 localized in cytoplasm and nucleus, the other fourproteins appear to be associated with the vacuolar membrane(Batistic et al. 2010). While vacuolar targeting of CBL2/3/6 isspecifically achieved by S-acylation of several cysteine resi-dues in the N-terminal tonoplast targeting sequence (Batisticet al. 2012;Tang et al. 2012),CBL10 probably reaches its mem-brane destination via the conventional secretory pathway(Kim et al. 2007), which is largely dependent on the putativeTMD in the N-terminus (Batistic et al. 2010). Similar to theirArabidopsis ortholog AtCBL10, both PtCBL10A andPtCBL10B contain a unique N-terminal extension sequencethat forms a single-pass hydrophobic domain. This putativetransmembrane span is required for subcellular localization ofPtCBL10s and targeting of PtCBL10-PtSOS2 complex to thetonoplast. It seems that proper targeting of PtCBL10s tocellular membranes is essential for the function in salt toler-ance because expression of the truncated soluble formPtCBL10ΔTMD cannot complement the cbl10 mutant (Sup-porting Information Fig. S4). Apart from being localized inthe vacuolar compartments,AtCBL10 was also reported to betargeted to the plasma membrane where it forms a signallingcomplex with SOS2 to regulate the Na+/H+ antiporter SOS1(Quan et al. 2007; Lin et al. 2009). In our subcellular localiza-tion studies on PtCBL10s, both PtCBL10A and PtCBL10Bresided in the vacuolar membranes of isolated poplar meso-phyll protoplasts whereas PtSOS3 was clearly present in theplasma membrane (Fig. 3). Consistently, PtCBL10s andPtSOS3 differentially targeted the common downstream com-ponent PtSOS2 to the tonoplast and plasma membrane,respectively (Fig. 6). Moreover, neither PtCBL10A norPtCBL10B could recruit PtSOS2 to the plasma membrane asefficiently as PtSOS3 did in the yeast RRS experiment(Fig. 5b).Based on these results,we speculate that PtCBL10Aand PtCBL10B serve as calcium sensor proteins mainly in thevacuolar membrane, especially when poplars are challengedwith high salt and osmotic stresses. However, we cannotexclude the possibility that PtCBL10s may also be associatedwith the plasma membrane in some specific cell types or undercertain physiological conditions.

In addition to their different subcellular localization,PtCBL10s and PtSOS3 display distinct expression patternsin poplars. The PtSOS3 gene is preferentially expressed inpoplar roots whereas the integral expression of PtCBL10s ismainly in aerial green tissues (Fig. 2). Besides, expression ofPtCBL10s but not PtSOS3 is slightly induced in response tosalinity (Fig. 2d; Tang et al. 2010), indicating that these twocalcium sensors are differentially regulated by salt stress atthe transcriptional level. More importantly, expression ofPtCBL10A and PtCBL10B but not PtSOS3 can rescue shootsensitivity of Arabidopsis cbl10 mutant to high salt condi-tions (Fig. 4), suggesting that PtCBL10A and PtCBL10B arefunctionally equivalent while PtSOS3 plays a distinct role inpoplar salt stress adaptation.This result is consistent with theearlier finding in Arabidopsis that SOS3 and SCaBP8/CBL10could not replace each other in the genetic complementation



test (Quan et al. 2007). It was further demonstrated thatCBL10 but not SOS3 is phosphorylated by SOS2 and thisspecific post-translational modification stabilizes SOS2-CBL10 interaction which is essential for the full function ofCBL10 (Lin et al. 2009). However, SOS3/CBL4 was recentlyshown to be phosphorylated by SOS2/CIPK24 as well(Hashimoto et al. 2012). Therefore, it is more reasonable toinfer that the spatial specificity of these two calcium sensorsmay contribute to their functional diversification in salt stressadaptation. In other words, CBL10-SOS2 and SOS3-SOS2complexes may have different targets in specific plant celltypes. This idea is supported by our transgenic poplarsoverexpressing PtCBL10s and PtSOS3. When overexpressedin poplars, even under the same control of constitutive35S promoter, PtCBL10s and PtSOS3 differentially regu-lated Na+ levels in different poplar tissues. In PtSOS3overexpression poplar, the Na+ content was greatly reducedin roots but increased in the stem during salt treatment(Fig. 8c). This is consistent with the putative role of PtSOS3-PtSOS2 in the activation of plasma membrane Na+/H+

antiporter PtSOS1. Indeed, SOS1 is not only responsible forextruding Na+ out of the root, but also critical for Na+ long-distance transport and partitioning between plant organs(Shi et al. 2002; Olías et al. 2009).That could also explain whyPtSOS3 overexpression poplar accumulates less Na+ in rootsbut more Na+ in shoots under normal conditions when thebasal Na+ is not harmful (Fig. 8a). Our data suggestedthat PtSOS3 might play a regulatory role in the long-distance transport of Na+ in poplar. On the other hand,overexpression of PtCBL10s lowered the leaf Na+ concentra-tions and retained Na+ in the stem under salt stress, and thusprevented Na+ accumulation from the photosynthetic tissues(Fig. 8c). This observation seems to conflict with the earlyhypothesis that CBL10 might play a role in Na+ sequestrationinto vacuole in Arabidopsis shoots (Kim et al. 2007).Vacuolarcompartmentalization of Na+ was believed to be mediated byNHX-type antiporters that play a critical role in plant salttolerance (Apse et al. 1999; Gaxiola et al. 1999; Quintero,Blatt & Pardo 2000). However, this notion has beenchallenged by recent genetic analyses of nhx mutants inArabidopsis. It was shown that nhx1 nhx2 double mutantswere specifically sensitive to external KCl but not to NaCl(Bassil et al. 2011; Barragán et al. 2012). Moreover, nhx1 nhx2mutant plants accumulated more Na+ in the shoot tissue thanthe WT in saline conditions, arguing the role of NHX1 andNHX2 in sequestering Na+ (Barragán et al. 2012). Transgenictomato plants overexpressing AtNHX1 had larger K+ vacu-olar pools but no enhancement of Na+ accumulation undersalt stress (Leidi et al. 2010). These results suggest thatNHX1/2 probably plays a major role in K+ homeostasisrather than in Na+ sequestration. In this regard, NHX1/2 maynot serve as a direct target for the CBL10-SOS2 complex.Further research will be directed to identify the elusiveCBL10 target(s) in the vacuolar membrane. Taken together,PtCBL10s and PtSOS3 probably fulfil distinct regulatoryfunctions in salt stress responses in poplar plants.

Poplars are widely grown as a ‘tree crop’ that producesplenty of wood, fibre and renewable biomass for energy.

Moreover, they can be utilized in environmental protectionas a protective barrier against soil erosion and a potentialphytoremediator in polluted soils. As most poplar cultivarsare very sensitive to salinity, it is necessary to develop salt-tolerant varieties. In spite of the complexity of plant salttolerance, overexpression of some key genes has beenshown to result in augmented salt tolerance (Yamaguchi &Blumwald 2005). In this regard, signalling components andregulatory genes are desirable candidates for the geneticengineering of salt-tolerant trees in that they may regulatemultiple downstream targets (Chinnusamy, Jagendorf & Zhu2005; Pardo 2010). Previously, overexpression of AtSOS3 orectopic expression of ZmCBL4 led to improved salt resist-ance in Arabidopsis (Wang et al. 2007; Yang et al. 2009).More recently, overexpression of tomato SlSOS2 or appleMdCIPK6L gene was independently reported to increasesalt tolerance of tomato plants (Huertas et al. 2012; Wanget al. 2012). However, such attempts have barely been madein developing transgenic trees. In the present work, we evalu-ated the feasibility of engineering PtCBL10s to improvesalt tolerance in poplar plants. Our results showed thatoverexpression of either PtCBL10s or PtSOS3 could main-tain a favourable K+/Na+ ratio in shoots or in roots, respec-tively, and thereby conferred enhanced salt tolerance ontransgenic aspens. The largely non-overlapping roles ofPtCBL10s and PtSOS3 in salt stress signalling make it pos-sible that co-overexpression of these calcium sensor proteinsmay further augment the tolerance of poplar plants to salin-ity stress. Alternatively, overexpression of their commondownstream kinase PtSOS2 would also provide a promisingapproach to superior salt-tolerant tree species.

ACKNOWLEDGMENTS

We thank Prof Yan Guo (China Agriculture University,Beijing) for providing the scabp8/cbl10 mutant seeds. Thiswork has been supported by the following grants: theNational Natural Science Foundation of China 31000120,31000288, 31171169, 31100212, 31270314, 31371228; theNational Basic Research Program of China 2010CB126600;the National Mega Project of GMO Crops 2013ZX08001003-007, 2013ZX08004002-006 and 2014ZX0800942B.

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Received 23 April 2013; received in revised form 19 July 2013; acceptedfor publication 26 July 2013

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:

Figure S1. Diagram of the advanced yellow fluorescentprotein (eYFP)-based BiFC vectors used in this study. Thep13SP-nYNT and p23SP-nYCT were constructed on thebackbone of pCambia-1300 and pCambia-2300, respectively.Expression cassettes and multiple cloning sites are shown inthe picture.Figure S2. Schematic representation of the constructs used tooverexpress PtCBL10A and PtCBL10B in poplar plants. ThePtCBL10A or PtCBL10B expression cassette is flanked bythe enhanced double cauliflower mosaic virus (2X CaMV)35S promoter and the octopine synthase (OCS) terminator.The vectors are built on the backbone of pCambia-1301 thatcontains GUS reporter gene as well as the HPTII selectiongene conferring hygromycin B resistance on transformedplants.Figure S3. Subcellular localization of PtCBL10-YFP andPtSOS3-YFP fusion proteins in Nicotiana benthamiana.Indicated constructs harbouring different YFP fusions wereexpressed in N. benthamiana leaves by Agrobacterium-mediated infiltration. The first panels on the left depict theYFP fluorescence in intact epidermal cells. The panels in thethird column show the YFP fluorescence in protoplasts pre-pared from the same leaf.The panels in the second and fourthcolumns are bright-field images.Figure S4. Expression of the truncated PtCBL10s cannotrescue the salt-sensitive phenotype of Arabidopsis cbl10.Four-day-old seedlings of WT, cbl10 and cbl10 expressingPtCBL10AΔTMD or PtCBL10BΔTMD transgene weretransferred to MS medium containing (a) 0, (b) 60 and (c)120 mm NaCl, respectively. The photographs were taken 10 dafter the initiation of the treatments. (d) Seedling freshweight was measured on the 10th day after the transfer.Error bars represent SE from triplicate experiments. Scalebars = 10 mm.Figure S5. Molecular confirmation of the stable transgeniclines overexpressing PtCBL10s in poplar plants. (a) PCRamplification of PtCBL10A or PtCBL10B transgenes withthe combination of 35S-specific and gene-specific primers. (b)Histochemical detection of GUS expression in the putativetransgenic poplar plants.Figure S6. Overexpression of PtSOS3 increases salt tolerancein transgenic poplar plants. (a) Growth of WT and two trans-genic lines L38, L314 in the absence of salt stress for 1 month.(b) Effects of 100 mm NaCl on the growth of WT, L38 andL314. Photographs were taken after 3 d of recovery fromthe salt treatment. Scale bars = 10 cm. (c) PCR and GUShistochemical analysis of the putative transgenic poplarplants overexpressing PtSOS3. (d) Quantitative RT-PCRanalysis of PtSOS3 expression in WT and different transgeniclines. (e–f) Measurements of plant height (e) and shootbiomass (f) after 3 d of recovery from 100 mm NaCl treat-ment. Values are means ± SD (n = 6) of two independentexperiments. Asterisks indicate significant difference in com-parison to WT (Student’s t-test, * P < 0.05).Table S1. Primers used in this study. This table lists the DNAsequence of the primers that have been used for isolatinggenes and cloning of the constructs in this study.



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