The woody plant poplar has a functionally conserved salt overlysensitive pathway in response to salinity stress

Ren-Jie Tang • Hua Liu • Yan Bao •

Qun-Dan Lv • Lei Yang • Hong-Xia Zhang

Received: 9 July 2010 / Accepted: 12 August 2010 / Published online: 29 August 2010

� Springer Science+Business Media B.V. 2010

Abstract In Arabidopsis thaliana, the salt overly sensi-

tive (SOS) pathway plays an essential role in maintaining

ion homeostasis and conferring salt tolerance. Here we

identified three SOS components in the woody plant Pop-

ulus trichocarpa, designated as PtSOS1, PtSOS2 and

PtSOS3. These putative SOS genes exhibited an overlap-

ping but distinct expression pattern in poplar plants and the

transcript levels of SOS1 and SOS2 were responsive to

salinity stress. In poplar mesophyll protoplasts, PtSOS1

was specifically localized in the plasma membrane,

whereas PtSOS2 was distributed throughout the cell, and

PtSOS3 was predominantly targeted to the plasma mem-

brane. Heterologous expression of PtSOS1, PtSOS2 and

PtSOS3 could rescue salt-sensitive phenotypes of the cor-

responding Arabidopsis sos mutants, demonstrating that

the Populus SOS proteins are functional homologues of

their Arabidopsis counterpart. In addition, PtSOS3 inter-

acted with, and recruited PtSOS2 to the plasma membrane

in yeast and in planta. Reconstitution of poplar SOS

pathway in yeast cells revealed that PtSOS2 and PtSOS3

acted coordinately to activate PtSOS1. Moreover, expres-

sion of the constitutively activated form of PtSOS2 par-

tially complemented the sos3 mutant but not sos1,

suggesting that PtSOS2 functions genetically downstream

of SOS3 and upstream of SOS1. These results indicate a

strong functional conservation of SOS pathway responsible

for salt stress signaling from herbaceous to woody plants.

Keywords SOS pathway � Salt stress � Populus �Arabidopsis � Functional conservation

Introduction

Soil salinity has become one of the major worldwide

agricultural problems. It is a severe limiting factor that

adversely affects plant growth in general and crop pro-

ductivity in particular. For most plants, high salt concen-

trations impose both an ionic and an osmotic stress (Zhu

2001), coupled with secondary stresses such as oxidative

stress (Borsani et al. 2001), genotoxicity (Albinsky et al.

1999) and nutritional disorders (Parida et al. 2004). Sodium

(Na?) toxicity represents the major ionic stress associated

with high salinity. The excessive accumulation of Na? in

the cytoplasm has injurious effects on plant cells in that it

prevents uptake of the essential mineral nutrient potassium

(K?), leading to insufficient cellular K? amount for

enzymatic reactions and osmotic adjustment. Therefore,

maintaining an optimal cytosolic K?/Na? homeostasis is

crucial for plant salt tolerance (Niu et al. 1995; Hasegawa

et al. 2000; Zhu 2003; Volkov et al. 2004). In order to

preserve a low Na? concentration in the cytoplasm, plants

have evolved several machineries at the cellular level

(Blumwald 2000; Horie and Schroeder 2004; Chen et al.

2007). One possible way is to restrict unidirectional Na?

uptake by roots (Rubio et al. 1995; Laurie et al. 2002).

Alternatively, Na? extrusion away from the cytoplasm (Shi

et al. 2002) and Na? compartmentation into the vacuole

(Apse et al. 1999) may serve as active means to minimize

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-010-9680-x) contains supplementarymaterial, which is available to authorized users.

R.-J. Tang � H. Liu � Y. Bao � Q.-D. Lv � L. Yang �H.-X. Zhang (&)

National Key Laboratory of Plant Molecular Genetics,

Institute of Plant Physiology and Ecology, Shanghai Institutes

for Biological Sciences, Chinese Academy of Sciences,

300 Fenglin Road, 200032 Shanghai, China

e-mail: hxzhang@sippe.ac.cn

123

Plant Mol Biol (2010) 74:367–380

DOI 10.1007/s11103-010-9680-x

sodium toxicity in plant cells. These vital Na? transloca-

tion processes are mediated by a battery of ion transporters

(Horie and Schroeder 2004) under the control of delicate

signaling networks that modulate their activities and

affinities (Xiong et al. 2002).

In Arabidopsis, the salt overly sensitive (SOS) pathway

is responsible for Na? homeostasis and salt tolerance under

salt stress. This pathway was established through genetic

identification of several salt overly sensitive (sos) mutants

followed by molecular and biochemical characterization

of the coding SOS proteins (Zhu 2002). A myristoylated

EF-hand calcium-binding protein encoded by SOS3 pre-

sumably senses and interprets the cellular calcium signal

elicited by salt stress (Liu and Zhu 1998; Ishitani et al.

2000). One of the primary downstream targets SOS2, a

serine/threonine protein kinase (Liu et al. 2000), is acti-

vated and recruited to plasma membrane via direct inter-

action with SOS3 (Halfter et al. 2000; Quintero et al.

2002). Subsequently, the SOS2-SOS3 complex phosphor-

ylates the Na?/H? antiporter SOS1 (Shi et al. 2000) to

stimulate its Na?/H? exchange activity at the plasma

membrane (Quintero et al. 2002; Qiu et al. 2002). In yeast,

co-expression of SOS2, SOS3, together with SOS1

increases salt tolerance of a yeast mutant deficient in

sodium transport more dramatically than expression of one

or two SOS proteins, suggesting that full activation of

SOS1 depends on the SOS2-SOS3 kinase complex (Quin-

tero et al. 2002). In Arabidopsis, vesicles of sos1, sos2 and

sos3 plants all display reduced plasma membrane Na?/H?

exchange activity compared to those of wild type plants,

and a constitutively activated SOS2 protein enhance the

Na?/H? exchange activity in a SOS1-dependent and

SOS3-independent manner (Qiu et al. 2002). Accordingly,

overexpression of SOS1 (Shi et al. 2003) or a constitutively

activated form of SOS2 (Guo et al. 2004) or the putative

upstream sensor SOS3 (Yang et al. 2009) all confers

enhanced salt tolerance on transgenic plants. Likewise, the

rice SOS1 homolog OsSOS1 functions as a plasma mem-

brane Na?/H? antiporter and is phosphorylated by the

SOS2–SOS3 kinase complex. Ectopic expression of

OsSOS1 suppressed the growth defects of Arabidopsis sos1

mutant upon salt treatment. The SOS2/SOS3 homolog in

rice, OsCIPK24/OsCBL4, acted coordinately to activate

OsSOS1 in yeast cells, and suppressed salt sensitivity of

Arabidopsis sos2/sos3 mutant, indicating that the SOS

pathway for salt tolerance is also operational in cereals

(Martinez-Atienza et al. 2007).

Although previous studies have highlighted important

roles of SOS pathway in the dicot herbaceous plant Ara-

bidopsis and the monocot plant rice, little is known about

the conservation of this pathway in trees. Populus has

become an important model in woody plants (Jansson

and Douglas 2007), due to its worldwide distribution,

sequenced genome and relative ease of genetic manipula-

tion (Tuskan et al. 2006). Poplars have also received

increasing attention as a renewable source of biomass for

energy and short-fiber pulp for paper making (Luo and

Polle 2009; Pilate et al. 2002). Recently, a putative plasma

membrane Na?/H? antiporter PeSOS1 from Populus eu-

phratica, has been isolated and shown to partially suppress

the salt-sensitivity of the Escherichia coli mutant strain

EP432 (Wu et al. 2007), but analysis of its in planta

function is still lacking. In order to better understand the

molecular mechanism underlying salt tolerance in woody

plants, we isolated three genes coding for key components

in the SOS pathway from the sequenced poplar genotype

Populus trichocarpa. By molecular analysis and functional

characterization of these three PtSOS genes, we show that

the perennial woody plant poplar has a conserved SOS

pathway in response to salinity stress.

Materials and methods

Plant materials, growth conditions and stress treatment

Two poplar genotypes Populus trichocarpa and Populus

alba 9 P. Berolinensis cv. Yin-Zhong were used in this

study. Generally, in vitro grown plants were subcultured

every month by aseptically transferring shoot apices to the

fresh MS medium (Murashige and Skoog 1962) supple-

mented with 0.03 mg/L NAA for rooting. Three-week-old

micro-propagated plantlets were subjected to salt stress by

directly pouring 30 mL of 200 mM NaCl solution into the

tissue-culture containers. Plantlets were also transferred

into individual pots (volume 6 L) after 3-day acclimation

to ex vitro conditions, and grown in the greenhouse under

an 14-h photoperiod comprising natural daylight supple-

mented with lamps (Philips, 600 W), giving a minimum

quantum flux density of 150 lEm-2s-1. The temperature

was kept at about 21–24�C in the daytime and 15–18�C at

night. All the plants were well irrigated according to

evaporation demands during different growth stages and

watered with half strength of Hoagland nutrient solutions

every other week. After 6 months’ growth, the trees would

reach a height of approximately 240 cm and a diameter of

1.2 cm. Various tissues were isolated from vigorously

growing trees including apical buds, young leaves, mature

leaves, elongating internodes, stem segments and roots.

Xylem and phloem tissues were simply separated by

stripping off the bark with a sharp blade. The samples were

frozen in liquid nitrogen immediately and stored at -80�C.

Arabidopsis thaliana plants were grown in the green-

house under long-day conditions (16 h light/8 h dark) at

the temperature 22–23�C. Sterilized seeds were plated on

MS medium solidified with 0.8% agar. All of the sos1, sos2

368 Plant Mol Biol (2010) 74:367–380

123

and sos3 mutants in this study were in the Col-0 gl1

background.

Reverse transcription PCR and quantitative real-time

PCR analyses

Total RNA was extracted with the RNAiso Reagent

(Takara, Japan) from different organs or tissues of two

types of poplar plants at the half-year growth stage in the

green house. Following the manufacturer’s instruction,

RNA samples were obtained with the same reagent from

the salt-treated poplar plantlets and 10-day old Arabidopsis

seedlings. After being treated with DNase I (Promega),

2 lg of total RNA was subjected to reverse transcription

reaction using the reverse transcriptase ReverTra Ace

(TOYOBO, Japan) at 42�C for 1 h. The resulting cDNA

was then used for PCR amplification with the gene-specific

primers listed below. For PtSOS1, the primers S1RT-F

(50-AAAGCCCATGGTTTCCAAGATG-30), and S1RT-R

(50-CCGC-TTCAAATGCTGCAATATC-30) were used;

For PtSOS2, the primers S2RT-F (50-AAAGCCCATGGTT

TCCAAGATG-30), and S2RT-R (50-AGTCATTGTTCGA

AGCAGGCAG-30) were used; For PtSOS3, the primers

S3RT-F (50-TGGAAAAATCGATCCTGACGAGTG-30),and S3RT-R (50-GCTGGTTTAAAATGCACGGATCAC-30)were used; For EF1b, the primers EF1b-F (50-GACAA

GAAGGCAGCGGAGGAGAG-30), and EF1b-R (50-CAA

TGAGGGAATCCACTGACACAAG-30) were used; For

ACTIN2, the primers ACT2-F (50-GGAAGGATCTGTAC

GGTAAC-30) and ACT2-R (50-GGACCTGCCTCATCA

TACT-30) were used.

Quantitative real-time PCR analysis was performed with

the RotorGene 3000 system (Corbett Research) using the

SYBR Green Realtime PCR Master Mix (TOYOBO,

Japan) to monitor double-stranded DNA products. Data

analysis was performed with Rotor-Gene software version

6.0 and relative amounts of mRNA were calculated based

on the comparative threshold cycle method. The relative

expression of each target gene was double-normalized

using the housekeeping gene EF1b and using the control

expression values measured at 0 h.

Subcellular localization of PtSOS1, PtSOS2

and PtSOS3

To determine the subcellular localization of PtSOS pro-

teins, stop-codon-less coding region of PtSOS1, PtSOS2 or

PtSOS3 was in-frame fused upstream to the YFP sequence

in the pA7-YFP vector, respectively. The resulting con-

structs containing PtSOS-YFP translational fusions were

transfected into poplar mesophyll protoplasts, essentially as

described by Sheen and colleges (Sheen 2001; Yoo et al.

2007). Fluorescence of YFP in the transformed protoplasts

was imagined using a confocal laser scanning microscope

(LSM510, Carl Zeiss) after the protoplasts were incubated

at 23�C for 16 h.

Yeast experiments

Yeast two hybrid assays were based on Matchmaker GAL4

Two-Hybrid System 3 (Clontech). The PtSOS2 coding

sequence was excised from the pBluescript II KS construct

using EcoRI-XhoI double digestion and subcloned into to

pGADT7 vector to generate an in-frame fusion with AD.

Similarly, PtSOS3 was inserted into pGBDT7 vector

through EcoRI-SalI sites to generate an in-frame fusion

with BD. To generate PtSOS2 devoid of the FISL motif,

inverse PCR-based mutagenesis was carried out on double-

stranded pKS-PtSOS2 plasmid, resulting in pKS-

PtSOS2DF. Then PtSOS2DF was subcloned into pGADT7

vector. These constructs and empty vector controls were

transformed into yeast strain AH109 by the PEG/LiAc

method. Yeast cells were plated onto SD/-Ade/-His/-Trp/-

Leu medium for stringent screening of the possible interac-

tions. b-galactosidase assays were performed as described in

the Clontech Yeast Protocols Handbook.

For experiments with the yeast Ras recruitment system

(RRS), the p426GPD-RAS vector was used to express RAS

fusion baits and the p425GPD vector (Mumberg et al.

1995) was used to express prey proteins. For p426GPD-

RAS construction, a Ras fragment without the stop codon

was produced by PCR amplification using the pYES-RRS

plasmid (Broder et al. 1998) as a template with the primers

RAS-F (50-CCCACTAGTATGACGGAATATAAGCTGG-30)and RAS-R (50-GGGGGATCC CTTGCAGCTCATGCAG

C-30), and then cloned into the p426GPD vector via SpeI-

BamHI double digestion. Afterwards, the coding sequence

of PtSOS2, PtSOSDF or AtSOS2 was in-frame fused

downstream to RAS in the p426GPD-RAS construct

between BamHI and SalI sites. PtSOS3 or AtSOS3 was

directly inserted into the p425GPD construct. Several

combinations of these plasmids and empty vector controls

were transferred into the yeast strain cdc25-2 (MATa, ade2,

his3, leu2, lys2, trp1, ura3, cdc25-2) and transformed yeast

cells were selected on SD/-Ura/-Leu medium. For inter-

action analysis, serial decimal dilutions of yeast cultures

(stating from OD = 1.0) were spotted onto YPD plates at

the permissive (24�C) or the restrictive (36�C) temperature.

Five randomly selected yeast colonies after transformation

were subjected to this assay.

To express SOS genes in yeast, PtSOS1 or AtSOS1 was

constructed into the p426PMA vector, a derivant from

p426GPD in which the GPD promoter was replaced by the

PMA1 promoter (Capieaux et al. 1989). The yeast vector

p414GPD (Mumberg et al. 1995) was employed to express

PtSOS2 or PtSOS3 alone. To coordinate expression of

Plant Mol Biol (2010) 74:367–380 369

123

PtSOS2 and PtSOS3 within a single plasmid, we con-

structed PtSOS2:CYC1-GPD:PtSOS3 on the backbone of

pBluescript II KS plasmid by successively introducing four

PCR fragments (the PtSOS2 CDS; the CYC1 terminator;

the GPD promoter; the PtSOS3 CDS) into the multiple

cloning site (MCS). The quadruple cassette was then sub-

cloned into the p414GPD plasmid via BamHI-XhoI double

digestion, resulting in the final construct harboring two

expression cassettes in tandem (GPD:PtSOS2:CYC1-

GPD:PtSOS3:CYC1). The Saccharomyces cerevisiae strain

B31 (MATa, ade2, can1, his3, leu2, trp1, ura3, mall0,

Dena1::HIS3::ena4, Dnha1::LEU2, Banuelos et al. 1998)

was transformed with one of or combinations of these

expression constructs. Na? tolerance tests were performed

in arginine-phosphate (AP) medium (8 mM phosphoric

acid, 10 mM L-Arg, 2 mM MgSO4, 0.2 mM CaCl2, 2%

glucose, plus vitamins and trace elements, pH = 6.0).

Aliquots (3 lL) were spotted onto AP plates supplemented

with 0, 120 and 240 mM NaCl in addition to 1 mM KCl,

and grown for 60 h at 30�C.

Bimolecular fluorescence complementation

(BiFC) assays

For generation of the BiFC vectors, the coding region of

PtSOS2 was subcloned via BamHI-SmaI into pSPYNE-

35S, resulting in PtSOS2YFPN; and the coding region of

PtSOS3 was cloned via BamHI-XhoI into pSPYCE-35S,

resulting in PtSOS3YFPC. Infiltration of Nicotiana benth-

amiana leaves was performed as previously described by

Walter et al. (2004). Protoplasts were prepared 3 days after

infiltration by cutting leaf discs into small pieces and

incubating for 3 h in the enzyme solution (0.4 M mannitol,

20 mM MES-K, 10 mM CaCl2, 5 mM b-mercaptoethanol,

0.1% BSA, 1% cellulase R10, 0.3% macerozyme R10, pH

5.7). Fluorescence of YFP in the leaf epidermal cells or

isolated protoplasts was imagined by a confocal laser

scanning microscope (LSM510, Carl Zeiss).

Arabidopsis transformation and complementation test

To prepare constructs for plant transformation, the open

reading frame of PtSOS1, PtSOS2 or PtSOS3 was digested

from the pBluescript II KS vectors with SmaI and SalI,

respectively. To produce the constitutively activated form

of PtSOS2, a Thr169-to-Asp mutation was introduced into

pKS-PtSOS2 by PCR-based Site-Directed Mutagenesis Kit

(Stratagene) using the primers 50-TTGGACTTCTTCATG

ACACATGTGGAACCCCG-30 and 50-CCCCTTCTGCGG

CAACGCACTCAATCC-30. The yielding pKS-PtSOS2TD

plasmid was also digested with BamHI and SalI to release

PtSOS2TD coding sequence. All the fragments were then

respectively inserted into a modified pCAMBIA-2301

vector downstream of the cauliflower mosaic virus 35S

promoter. These plasmids were separately introduced into

the Agrobacterium GV3101 strain for Arabidopsis trans-

formation. Putative transgenic plants were screened on MS

medium containing 50 lg/L kanamycin and transferred to

soil for propagations. Kanamycin-resistant plants of T2

generation were selected and subjected to transgene

expression analyses. Seeds of homozygous T2 lines were

harvested and the T3 generation was employed for com-

plementation tests on MS agar plates supplemented with

NaCl, as indicted for each case.

Results

Isolation of PtSOS1, PtSOS2 and PtSOS3 from Populus

We performed homology-based BLAST searches (Altschul

et al. 1997) to collect candidate gene sequences coding for

poplar SOS pathway components from the Joint Genome

Initiative poplar database (JGI, Populus trichocarpa

genome portal v1.1; http://genome.jgi-psf.org/Poptr

1_1/Poptr1_1.home.html). Two SOS1 (with 89% identity),

SOS2 (with 91% identity) and SOS3 (with 94% identity)

homologues were identified from the whole Populus gen-

ome. Due to high similarity within individual Populus SOS

candidates, we chose only one gene of each as a repre-

sentative, with the closest homology to Arabidopsis SOS

counterpart, for subsequent studies. These three genes were

designated as PtSOS1, PtSOS2 and PtSOS3. Total RNA

was extracted from salt-treated plantlets of Populus

trichocarpa and first-strand cDNA was used to amplify the

coding region of each gene on the basis of public genomic

sequence. The amplified fragments were subcloned to

pBluescript II KS vector and then sequenced independently

three times to confirm proper removal of introns and

fidelity of the polymerase. After manual revision of the

isolated CDS and its coding protein, sequence alignment

was conducted between PtSOS1, 2, 3 and AtSOS1, 2, 3

respectively (Figs. S1, S2, S3). The three putative SOS

proteins in poplar bear significant resemblance to their

Arabidopsis counterparts in size and structure.

The poplar SOS components show overlapping

but distinct expression pattern

As the first step to characterize SOS pathway in woody

plants, expression of poplar SOS genes was analyzed to

clarify whether the three components co-localize and

thereby co-function in planta. We performed RT–PCR to

determine relative transcript abundance in various poplar

370 Plant Mol Biol (2010) 74:367–380

123

tissues from two genotypes in our lab, i.e. the sequenced

genotype Populus trichocarpa and an aspen genotype

Populus alba 9 P. Berolinensis (a common cultivar in the

North of China). The final amount of EF1b products ver-

ified that all samples contained approximately equal

amount of cDNA. In Populus trichocarpa, PtSOS1 and

PtSOS2 exhibited generally high, uniform and ubiquitous

expression in all tissues (Fig. 1a). Strong expression of

PtSOS1 was detected in root, juvenile leaf, stem, xylem

and apex while expression of PtSOS2 might be slightly

higher in young green tissues such as juvenile leaf and apex

than in other organs. The transcript of PtSOS3 was low in

most poplar tissues but relatively abundant in root. In

Populus alba 9 P. Berolinensis, a very similar expression

pattern was observed with regard to PabSOS1 and Pab-

SOS2, while preferential expression of PabSOS3 in root

tissues was even more evident (Fig. 1b). In conclusion, the

poplar SOS components show an overlapping but also a

distinct expression pattern, which may be analogous among

different Populus species.

Expression of SOS genes in the poplar is regulated

by salinity stress

To further examine whether the expression of poplar

SOS components is modified by salinity stress, 3-week-old

micro-propagated poplar plantlets were subjected to

200 mM NaCl for increasing exposure periods and the

transcript level of SOS1, SOS2 and SOS3 was analyzed by

quantitative real-time PCR. Since the sequenced genotype

Populus trichocarpa is extremely susceptible to salinity and

became wilted during our treatment period, we employed

Populus alba 9 P. Berolinensis in this test as our experi-

mental materials. In roots, PabSOS1 was rapidly induced

after the onset of salt stress and increased up to the maximal

level (around fivefold) at the twelfth hour during the NaCl

treatment. Expression of PabSOS2 coordinately increased

within 3 h, reached a maximal threefold to fourfold induc-

tion between 6 and 12 h, and thereafter declined. In contrast,

expression of PabSOS3 in roots was hardly responsive to

salinity stress during our experiment (Fig. 2a). In shoot

tissues (Fig. 2b), transcripts of both PabSOS1 and PabSOS2

began to accumulate within the first 3 h of NaCl treatment

and retained continuously higher expression than basal level

from 6 to 24 h. However, the transcript level of PabSOS3 in

above-ground tissues was reduced almost by half upon 1-h

salt treatment and recovered to the near-basal level after

12 h of salt treatment. These results indicate that SOS1 and

SOS2, but not SOS3, are salt responsive genes in the poplar

SOS pathway.

Subcellular localization of PtSOS1, PtSOS2

and PtSOS3 in poplar cells

Proteins that participate in the same pathway are supposed

to occupy overlapped locations within the cell. In order to

investigate subcellular localization of the Populus SOS

components in plant cells, we fused PtSOS1, PtSOS2 and

PtSOS3 in-frame to the N-terminus of yellow fluorescent

protein (YFP), respectively. The chimeric construct was

delivered into mesophyll protoplasts prepared from poplar

leaf tissues by the polyethylene glycol transformation

method (Sheen 2001; Yoo et al. 2007). Each fusion protein

was transiently expressed under the control of the consti-

tutive 35S promoter and its subcellular localization was

visualized by confocal microscopy. As expected in the case

of the positive control YFP alone, a diffuse green fluores-

cence typical of cytosolic location was observed with a

tendency to accumulate in the nucleus and near the plasma

membrane (Fig. 3, top panels). When fused to YFP,

PtSOS1 was exclusively localized in the plasma membrane

of poplar protoplasts (Fig. 3, second panels from top),

consistent with its identity as a Na?/H? exchanger at the

cell membrane. PtSOS2-YFP, however, was dispersed

Fig. 1 Expression analysis of the SOS1, 2, 3 genes in different

tissues of Populus trichocarpa (a) and Populus alba 9 P. Berolin-ensis (b). cDNA derived from the indicated tissues was used as a

template for PCR with the gene specific primers at different numbers

of PCR cycles. Amplification of the genomic DNA sample as a

control template was also carried out to rule out its possible

contamination in each cDNA sample. PCR products were visualized

by agarose gel electrophoresis and ethidium bromide staining. The

elongation factor gene EF1b was employed as an internal control

Plant Mol Biol (2010) 74:367–380 371

123

throughout the cytoplasm, within the nucleus and also at

the cell periphery, quite similar to YFP alone (Fig. 3, third

panels from top). PtSOS3-YFP was predominantly targeted

to the plasma membrane with leaky intracellular localiza-

tion (Fig. 3, bottom panels). These observations are in

favor of the physical possibility that PtSOS1, PtSOS2 and

PtSOS3 could be involved in the same pathway at the

plasma membrane of poplar cells.

Populus SOS proteins are functional homologues

of their Arabidopsis counterparts

The Arabidopsis mutants in the SOS pathway, i.e. sos1,

sos2 and sos3, are specifically hypersensitive to high

external Na? concentrations. To dissect the biological

function of PtSOS proteins, we constructed transgenic

Arabidopsis lines heterologously expressing each PtSOS

gene in the corresponding sos mutant background for

functional complementation tests. We selected four inde-

pendent complemented lines from rescued sos1, sos2 and

sos3 mutants, respectively. By RT–PCR analysis, we

confirmed the expression of PtSOS1 transgene in sos1,

PtSOS2 transgene in sos2, and PtSOS3 transgene in sos3

(Fig. 4a). Preliminary assays on salt tolerance of these

transgenic lines for each gene showed that they had similar

salt tolerance capacity, and therefore two representative

transgenic lines (CS12, CS16; CS26, CS29; CS35, CS36)

of each gene were chosen for subsequent phenotypic

analyses. In the absence of NaCl treatment, wild type

plants, sos mutants and PtSOS complemented lines dis-

played relatively uniform growth phenotype, although

roots of the seedlings in sos1 background were slightly

shorter (Fig. 4b, top panels). When treated with 60 mM

NaCl, growth of sos1, sos2 and sos3 mutants were all

inhibited compared with wild type plants, albeit to different

extent. Expression of PtSOS1 in sos1, PtSOS2 in sos2 and

PtSOS3 in sos3 substantially alleviate the growth retarda-

tion imposed by moderate salt stress (Fig. 4b, middle

panels). Under 120 mM NaCl treatment, growth inhibition

of sos2 and sos3 mutants was even more pronounced.

Fig. 2 Quantitative real-time

PCR analyses of poplar SOS

components in response to salt

stress. Three-week-old poplar

plantlets grown on MS medium

were treated with 200 mM

NaCl. Samples were collected at

0, 1, 3, 6, 12 and 24 h after the

initiation of treatment. Total

RNA was extracted from roots

a and shoots b respectively, and

real-time PCR analyses were

performed with SOS1, 2 and 3gene specific primers. The

relative expression of each

target gene was double-

normalized using the

housekeeping gene EF1b and

using the control expression

values measured at 0 h. Data

represents the average of three

independent experiments ±SD

372 Plant Mol Biol (2010) 74:367–380

123

Seedlings of sos1 were killed by this high concentration of

salt stress. Nevertheless, the sos1 mutants expressing

PtSOS1 could survive at this lethal NaCl level. The salt-

sensitive phenotype was also greatly suppressed in

PtSOS2- and PtSOS3-complemented lines (Fig. 4b, bottom

panels). A detailed analysis of root length and seedling

fresh weight showed that all the rescued transgenic plants

by PtSOS genes were generally restored to a near-wild-type

level of salt tolerance, although with differential extents

between different lines (Fig. 4c–h). These data indicate the

poplar SOS proteins can substitute for the corresponding

Arabidopsis SOS components, thereby being functional

orthologues of their Arabidopsis counterparts.

PtSOS3 interacts with and recruits PtSOS2

to the plasma membrane

In Arabidopsis, SOS2 interacted with SOS3 to form a

kinase complex for proper biological functions (Halfter

et al. 2000; Gong et al. 2004). Here we determined the

physical interaction between PtSOS2 and PtSOS3 by yeast

two hybrid assays. A combination of the bait plasmid

pGBKT7-PtSOS3 with the prey construct pGADT7-SOS2

resulted in full transcriptional activation of HIS, ADE and

lacZ reporters, whereas co-transformation of either empty

vector with the construct pGBKT7-PtSOS3 or pGADT7-

SOS2 did not activate any of the reporter genes. The FISL

motif (also known as NAF domain) in the SOS2 regulatory

region is necessary and sufficient for SOS3 interaction

(Guo et al. 2001; Albrecht et al. 2001). Deletion of this

conserved FISL motif away from PtSOS2 protein

(PtSOS2DF) abolished its interaction with PtSOS3

(Fig. 5a), suggesting that PtSOS2 also interacts with

PtSOS3 via the FISL motif.

To further address spatial specificity of PtSOS2–PtSOS3

complex formation, we employed the Ras recruitment

system (RRS) to monitor membrane targeting of PtSOS2

by PtSOS3 interaction. This system is based on the ability

of a mammalian Ras variant to be localized to the plasma

membrane through the interaction between two proteins.

Ras membrane localization via protein–protein interaction

results in complementation of the yeast thermo-sensitive

mutant cdc25-2 deficient in the Ras guanyl nucleotide

exchange factor (Broder et al. 1998). As shown in Fig. 6b,

expression of RAS-PtSOS2 fusion alone without PtSOS3

failed to restore yeast growth at the restrictive temperature

(36�C), indicating a cytosolic localization. However, the

RAS-SOS2 fusion was efficiently recruited to the plasma

Fig. 3 Subcellular localization

of PtSOS1-, 2- and 3-YFP

fusion proteins. Confocal

microscopy analysis of YFP

signals from poplar mesophyll

protoplasts transiently

expressing YFP or PtSOS-YFP

fusions as indicated. Scalebar = 5 lm

Plant Mol Biol (2010) 74:367–380 373

123

membrane upon co-expression of PtSOS3, allowing cdc25-

2 strain to grow at 36�C. RAS fusion with the PtSOS2

mutant lacking FISL motif disrupted PtSOS3 interaction

and thus did not restore the thermo-tolerance in the pres-

ence of PtSOS3. In conclusion, PtSOS3 recruited PtSOS2

to the plasma membrane through their physical interaction.

374 Plant Mol Biol (2010) 74:367–380

123

We also investigated the interaction between PtSOS2

and PtSOS3 in planta using bimolecular fluorescence

complementation (BiFC) assays (Walter et al. 2004). The

BiFC results shown in Fig. 5c not only confirmed the in

vivo PtSOS2-PtSOS3 interaction but also clearly rein-

forced the evidence that specific interaction of the two

proteins take place at or near the plasma membrane of plant

cells.

Reconstitution of the poplar SOS pathway in yeast cells

In yeast Saccharomyces cerevisiae, the Na?-pumping

ATPase ENA1-4 and the Na?/H? antiporter NHA1 are two

major sodium efflux proteins at the plasma membrane

responsible for most of the sodium extrusion. The B31

strain devoid of these two Na? transporters was defective

in sodium efflux and extremely sensitive to high external

Na? concentrations (Banuelos et al. 1998; Kinclova et al.

2001). To test whether the putative Na?/H? antiporter

PtSOS1 could be activated by PtSOS2–PtSOS3 complex,

we reconstituted poplar SOS regulatory pathway in yeast

cells by expressing different combinations of PtSOS

Fig. 5 Interaction between PtSOS2 and PtSOS3 in yeast and plant

cells. (a) Yeast strain AH109 harboring BD-PtSOS3 fusion in

pGBKT7 vector (or pGBKT7 empty vector) was transformed with

AD-PtSOS2, PtSOS2DF or PtSOS2TD fusion in pGADT7 vector (or

pGADT7 empty vector). The interaction was monitored by the

ability of yeast transformants to grow on selective medium minus

Leu, Trp, His and Ade, or by b-galactosidase assay on a piece of

filter paper. (b) Recruitment of PtSOS2 to the plasma membrane by

PtSOS3. The yeast cdc25-2 mutant was transformed with the plasmid

combinations as indicated on the left. Three microliters of serial

decimal dilutions were spotted onto YPD plates at the permissive

(24�C) or at the restrictive (36�C) temperature. Cells expressing

RAS-PtSOS2 in combination with PtSOS3 or RAS-AtSOS2 with

AtSOS3 combination were able to grow at 36�C, indicating

interaction between the two proteins at the plasma membrane.

(c) PtSOS2 interacted with PtSOS3 in planta. Empty YFP construct

(as a positive control) or different combinations of the N-YFP and

C-YFP fusion constructs as indicated were co-transfected into the

tobacco (N. benthamiana) leaves. The images were collected under

the Zeiss confocal microscope. The left panels depict the fluores-

cence in intact leaf epidermal cells (together with bright-field images

on the right), and the right panels exhibit the fluorescence signals in

protoplasts prepared from the same leaf (together with bright-field

images on the right)

Fig. 6 Reconstitution of poplar SOS pathway in yeast cells. The

yeast B31 (Dena1-4, Dnha1) strain transformed with an empty vector

or expressing the indicated combination of Populus SOS genes were

grown overnight in liquid selective medium. Three microliters of

serial decimal dilutions were spotted onto plates of arginine

phosphate (AP) medium with 1 mM KCl and supplemented with 0,

120 and 240 mM NaCl. Plates were incubated at 30�C for 60 h. The

Arabidopsis SOS1 gene AtSOS1 was used as a positive control.

Plasmids used for expression of SOS genes were described in

‘‘Materials and methods’’

Fig. 4 Functional complementation of Arabidopsis sos1, sos2 and

sos3 mutants by poplar PtSOS1, PtSOS2 and PtSOS3, respectively.

a Expression of PtSOS1, 2, 3 in each sos mutant as indicated. RT–

PCR products were amplified from seedlings of wild type, sosmutants and four corresponding complementary lines. The Arabid-opsis ACTIN2 gene was used as an internal control. b Four-day-old

seedlings of wild-type, sos mutants and complementary lines CS12,

CS16, CS26, CS29, CS35, CS36 were transferred to MS medium

containing 0, 60 and 120 mM NaCl, respectively. The pictures were

taken after 8 days of treatment. Seedling fresh weight c–e and root

length f–h were measured at day 8 after the transfer. Error barsrepresent SD (n = 10)

b

Plant Mol Biol (2010) 74:367–380 375

123

proteins in the B31 background. As depicted in Fig. 6,

expression of PtSOS1 alone only marginally elevated

halotolerance of yeast cells. Additional expression of

PtSOS2, but not PtSOS3, enabled the yeast strain to survive

better under moderate salinity stress (120 mM NaCl),

above levels imparted by SOS1 alone, but still failed to

afford yeast growth in high saline condition (240 mM

NaCl). Concurrent expression of three poplar SOS com-

ponents PtSOS1, PtSOS2 and PtSOS3 dramatically aug-

mented the salt tolerance of yeast cells, capable to sustain

growth in AP medium containing up to 240 mM NaCl.

This enhancement in salt tolerance was observed only in

the presence of PtSOS1, excluding the possibility that the

PtSOS2–PtSOS3 complex was unmasking endogenous

yeast activity. PtSOS2–PtSOS3 complex was also able to

activate AtSOS1 (as a positive control in our experiment),

with even greater efficacy, suggesting that the regulation of

SOS1 activity might be evolutionally conserved between

woody and herbaceous plant species.

Expression of Constitutively Active PtSOS2 (PtSOS2TD)

complemented salt-sensitive phenotype of sos2 mutant

entirely, and that of sos3 partially, but did not rescue salt

hypersensitivity of sos1 at all.

Physical interaction and yeast reconstitution experi-

ments in this study have demonstrated that the putative

poplar SOS components should be working in the same

pathway as those in Arabidopsis. To further consolidate

this notion in planta, we tested whether PtSOS2 functions

dependent of SOS1 and downstream of SOS3 in salt

tolerance. We generated a constitutively active form of

PtSOS2 (designated as PtSOS2TD) by mutating the 169th

amino acid threonine (T) to aspirate (D) in the activation

loop of this kinase (Fig. S2). This single-point mutation in

SOS2 substantially increased the kinase activity (Guo

et al. 2001) but did not affect its interaction with SOS3

(Fig. 5a), resulting in a constitutively activated and

functional variant of SOS2 both in vitro and in vivo (Guo

et al. 2004). The Arabidopsis sos1, sos2 and sos3 mutants

were transformed with PtSOS2TD under the control of

cauliflower mosaic virus 35S promoter. By RT–PCR

assay using PtSOS2 specific primers, we identified 10–15

transgenic lines overexpressing PtSOS2TD in sos1, sos2

or sos3 mutant background and selected two representa-

tive lines of each exhibiting similar expression levels for

subsequent phenotypic analysis (Fig. 7a). Expression of

PtSOS2TD in sos1 background failed to rescue its salt

overly sensitive phenotype, as is shown in Fig. 7b. The

transgenic sos1 plants and sos1 parental mutants dis-

played equally severe growth defect on MS plates sup-

plemented with 120 mM NaCl (Fig. 7c, f). However,

transgenic sos2 plants harboring PtSOS2TD grew like the

wild-type plants upon high salt treatment (Fig. 7b, middle

panels). A statistical analysis of root length (Fig. 7d) and

fresh weight (Fig. 7g) further indicated that salt hyper-

sensitivity of sos2 was fully rescued by constitutive

expression of the poplar activated form of SOS2. When

expressed in sos3 background, PtSOS2TD partially sup-

pressed the salt-sensitive phenotype of sos3 mutants

(Fig. 7b, right panels). The sos3 transformants harboring

PtSOS2TD grew bigger than sos3 mutants on saline

medium, as revealed by partially recovered fresh weight

(Fig. 7h) and less inhibited root length (Fig. 7e). Taken

together, the enhanced activity or functionality of PtSOS2

is not sufficient for salt tolerance in the absence of SOS1

but somewhat bypass the SOS3 deficiency in planta. By

virtue of strong conservation between Populus and Ara-

bidopsis SOS proteins, the functional complementation

tests here could imply that in poplar trees, PtSOS2 might

function downstream of PtSOS3 and upstream of PtSOS1,

as well.

Discussion

Adaptation to high salinity is one of the important concerns

for woody perennials during their lifespan and over evo-

lutionary timescales. The molecular mechanisms underly-

ing salt tolerance and salt stress signaling are poorly known

in woody plants. With the completion of genome

sequencing in Populus trichocarpa (Tuskan et al. 2006)

and rapidly developing genomic tools, a handful of genes

related to salt stress tolerance (Ottow et al. 2005; Wu et al.

2007; Wang et al. 2008; Martin et al. 2009; Chen et al.

2009; Ye et al. 2009) were isolated and characterized from

the genus Populus that has become the accepted model for

perennial trees (Jansson and Douglas 2007). However,

most of those studies were focused on the biological

function of individual genes, without interpretation of a

gene regulatory network leading to fine-tuning of adaptive

processes. In this report, for the first time, we characterized

the Populus SOS pathway by integrated analysis of three

component genes. Several lines of molecular evidence

support the conclusion that poplar plants have a functional

salt overly sensitive pathway orthologous to the well-doc-

umented Arabidopsis SOS pathway. First, the putative

PtSOS components share high similarity with AtSOS pro-

teins in structure implying their similar biochemical

properties. Second, the overlap in expression and subcel-

lular localization of three poplar SOS elements provides a

prerequisite for their coordinated actions in the same

pathway governing salt stress adaptation. Third, the poplar

SOS orthologues can substitute for the endogenous Ara-

bidopsis counterparts revealed by functional

376 Plant Mol Biol (2010) 74:367–380

123

complementation tests in sos mutants. Above all, PtSOS3

recruited PtSOS2 to the plasma membrane via direct

physical interaction and the PtSOS2–PtSOS3 complex

activates PtSOS1 in yeast cells.

In Populus, although the expression of poplar SOS genes

is generally ubiquitous and largely overlapping, each gene

may exhibit a distinct expression pattern to some extent.

For example, the expression of PtSOS1 in root and in shoot

Fig. 7 Expression of a constitutively activated poplar SOS2 version

(PtSOS2TD) in Arabidopsis sos mutants. a Transgene expression of

PtSOS2TD (the 169 amino acid Thr was mutated to Asp) in sos1(LS11, LS12), sos2 (LS21, LS22) and sos3 (LS31, LS32) background.

The Arabidopsis ACTIN2 gene was used as an internal control.

b Four-day-old seedlings of wild-type, sos mutants and selected

transgenic lines in different sos mutant background were transferred

to MS medium containing 0 or 120 mM NaCl, respectively. The

pictures were taken after 10 days of treatment. Seedling fresh weight

c–e and root length f–h were measured at day 10 after the transfer.

Error bars represent SD (n = 10)

Plant Mol Biol (2010) 74:367–380 377

123

was comparable, whereas PtSOS2 was preferentially

expressed in green tissues rather than roots (Fig. 1). In

Populus alba 9 P. Berolinensis, the expression of Pab-

SOS1 and PabSOS2 was rapidly induced upon salt treat-

ment, with a differential manner in root and in shoot

(Fig. 2). It seemed that the salt-induced up-regulation of

PabSOS1 and PabSOS2 had a long-term effect in shoots

with moderate levels, but was more responsive and tran-

sient in roots. The simultaneous induction of PabSOS1 and

PabSOS2 transcripts by salinity also suggested a fine-tuned

regulation of SOS elements at the gene expression level.

However, the expression of PabSOS3, the upstream com-

ponent of SOS pathway, was not responsive to salt treat-

ment in our test, either in root (Fig. 2a) or in shoot

(Fig. 2b), presumably because SOS3 senses calcium sig-

nals and triggers downstream signaling primarily at protein

modification but not the transcription level. We also

noticed that the transcript abundance of PtSOS3 was pretty

high in root but remarkably diminished in the aerial parts

(Fig. 1). This is consistent with the root-specific expression

of AtSOS3 in Arabidopsis (Quan et al. 2007). On the other

hand, the SOS3-like calcium binding protein 8 (SCaBP8)/

CBL10 was recently identified as a shoot-specific player in

the SOS pathway (Quan et al. 2007). Like SOS3, it

recruited SOS2 to the plasma membrane, but uniquely

underwent phosphorylation by SOS2, leading to stabilized

SCaBP8–SOS2 complex for SOS1 activation (Lin et al.

2009). Interestingly, we found a SCaBP8 homolog existing

in Populus genome to be predominantly expressed in the

above-ground tissues (unpublished data). This fact may

implicate a tissue-differential regulation of salt response in

the poplar SOS pathway.

The sodium efflux protein SOS1 functions as a pivotal

effector molecule in the SOS pathway, and it has been

suggested to fulfill several facets of important roles per-

taining to salt tolerance. To begin with, at the cellular level,

SOS1 would act extruding the excessive Na? to prevent Na?

accumulation in the cytoplasm, as it has been shown in

Arabidopsis (Qiu et al. 2003), rice (Martinez-Atienza et al.

2007), wheat (Xu et al. 2008), Thellungiella (Oh et al. 2009)

and tomato (Olıas et al. 2009). In Populus, the salt-tolerant

genotype Populus euphratica exhibited a higher transcript

abundance of genes related to Na?/H? antiport compared to

the salt-sensitive P. popularis (Ding et al. 2010). In a recent

study based on the scanning ion-selective electrode tech-

nique, it was concluded that Na? extrusion in stressed

Populus euphratica roots was the result of an active Na?/H?

antiport across the plasma membrane (Sun et al. 2009),

which might be mediated by PeSOS1 reported previously

(Wu et al. 2007). In addition, at the whole-plant level, it was

suggested that SOS1 controls long-distance Na? transport

(Shi et al. 2002) and affects Na? partitioning between plant

organs (Olıas et al. 2009). These Na? distribution processes

would be particularly important for woody poplars because

of their big stature and long lifespan. A precise assessment

of possible contributions of PtSOS1 in this aspect awaits the

availability of RNAi lines and/or overexpressors of trans-

genic poplars. Furthermore, SOS1 takes part in the cross-

talk between the ion-homesotasis and oxidative-stress sig-

naling pathways. It was reported that stress-induced SOS1

mRNA stability in Arabidopsis was mediated by reactive

oxygen species (ROS) and SOS1 played negative roles in

tolerance to oxidative stress (Chung et al. 2008). SOS1

interacted with RCD1, a regulator of radical-based signal-

ing, through the long C-terminal cytoplasmic tail, further

suggesting its involvement in regulating oxidative-stress

responses (Katiyar-Agarwal et al. 2006). In NaCl-stressed

Populus euphratica cells, H2O2 production and cytosolic

Ca2? signals were integrated to mediate K?/Na? homeo-

stasis (Sun et al. 2010; Zhang et al. 2007). It needs further

experimentation how the poplar SOS pathway functions in

such signaling cross-talk. Last but not least, by affecting

endocytosis under salinity conditions, SOS1 may also play

dynamic roles in vacuole integrity, membrane trafficking, as

well as pH homeostasis (Oh et al. 2009; Oh et al. 2010).

Further work on poplar SOS genes will be instructed by and

focused on all the above findings so as to elucidate func-

tional significance of SOS pathway in woody species. On the

other hand, engineering PtSOS genes identified in this study

offer good opportunities towards generation of transgenic

tree species with improved salt tolerance, which may be

grown on saline land to prevent erosion and potentially to

ameliorate soils (Singh 1998). At present, genetic transfor-

mation of poplar cultivars by overexpressing the three

PtSOS components is under way in our laboratory.

Acknowledgments We thank Dr. Jorg Kudla (Institut fur Botanik,

Universitat Munster, Germany) for providing the pSPYNE-35S and

pSPYCE-35S plasmids. We are also grateful to Dr. Ami Aronheim for

RRS materials and Dr. Lydie Maresova for the B31 yeast strain.

Special thanks would go to Dr. Rongmin Zhao (University of Tor-

onto, Canada) for his technical guidance on yeast experiments. This

work was supported by the following grants: the National Natural

Science Foundation of China (30872044), the National Basic

Research Program of China (2006CB100106), the National mega

project of GMO crops (2008ZX08001-003; 2008ZX08004-002;

2008ZX08010-004); Shanghai Science & Technology Development

Fund (0853Z111C1; 08DZ2270800).

References

Albinsky D, Masson JE, Bogucki A, Afsar K, Vass I, Nagy F,

Paszkowski J (1999) Plant responses to genotoxic stress are

linked to an ABA/salinity signaling pathway. Plant J 17:73–82

Albrecht V, Ritz O, Linder S, Harter K, Kudla J (2001) The NAF

domain defines a novel protein-protein interaction module

conserved in Ca2?-regulated kinases. EMBO J 20:1051–1063

378 Plant Mol Biol (2010) 74:367–380

123

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,

Lipman DJ (1997) Gapped BLAST and PSI-BLAST, a new

generation of protein database search programs. Nucleic Acids

Res 25:3389–3402

Apse MP, Aharon GS, Snedden WS, Blumwald E (1999) Salt

tolerance conferred by overexpression of a vacuolar Na?/H?

antiport in Arabidopsis. Science 285:1256–1258

Banuelos MA, Sychrova H, Bleykasten-Grosshans C, Souciet JL,

Potier S (1998) The Nha1 antiporter of Saccharomyces cerevi-siae mediates sodium and potassium efflux. Microbiology

144:2749–2758

Blumwald E (2000) Sodium transport and salt tolerance in plants.

Curr Opin Cell Biol 12:431–434

Borsani O, Valpuesta V, Botella MA (2001) Evidence for a role of

salicylic acid in the oxidative damage generated by NaCl and

osmotic stress in Arabidopsis seedlings. Plant Physiol 126:

1024–1030

Broder YC, Katz S, Aronheim A (1998) The Ras recruitment system,

a novel approach to the study of protein–protein interactions.

Curr Biol 8:1121–1124

Capieaux E, Vignais ML, Setenac A, Goffeau A (1989) The yeast

H?-ATPase gene is controlled by the promoter binding factor

TUF. J Biol Chem 264:7437–7446

Chen Z, Pottosin II, Cuin TA, Fuglsang AT, Tester M, Jha D, Zepeda-

Jazo I, Zhou M, Palmgren MG, Newman IA, Shabala S (2007)

Root plasma membrane transporters controlling K?/Na? homeo-

stasis in salt stressed barley. Plant Physiol 145:1714–1725

Chen J, Xia X, Yin W (2009) Expression profiling and functional

characterization of a DREB2-type gene from Populus euphra-tica. Biochem Biophys Res Commun 378:483–487

Chung JS, Zhu JK, Bressan RA, Hasegawa PM, Shi H (2008)

Reactive oxygen species mediate Na?-induced SOS1 mRNA

stability in Arabidopsis. Plant J 53:554–565

Ding M, Hou P, Shen X, Wang M, Deng S, Sun J, Xiao F, Wang R,

Zhou X, Lu C, Zhang D, Zheng X, Hu Z, Chen S (2010) Salt-

induced expression of genes related to Na?/K? and ROS

homeostasis in leaves of salt-resistant and salt-sensitive poplar

species. Plant Mol Biol 73:251–269

Gong D, Guo Y, Schumaker KS, Zhu JK (2004) The SOS3 family of

calcium sensors and SOS2 family of protein kinases in

Arabidopsis. Plant Physiol 134:919–926

Guo Y, Halfter U, Ishitani M, Zhu JK (2001) Molecular character-

ization of functional domains in the protein kinase SOS2 that is

required for plant salt tolerance. Plant Cell 13:1383–1400

Guo Y, Qiu QS, Quintero FJ, Pardo JM, Ohta M, Zhang C,

Schumaker KS, Zhu JK (2004) Transgenic evaluation of

activated mutant alleles of SOS2 reveals a critical requirement

of its kinase activity and C-terminal regulatory domain for salt

tolerance in Arabidopsis. Plant Cell 16:435–449

Halfter U, Ishitani M, Zhu JK (2000) The Arabidopsis SOS2

protein kinase physically interacts with and is activated by the

calcium-binding protein SOS3. Proc Natl Acad Sci USA 97:

3735–3740

Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular

and molecular responses to high salinity. Annu Rev Plant Biol

51:463–499

Horie T, Schroeder JI (2004) Sodium transporters in plants. Diverse

genes and physiological functions. Plant Physiol 136:2457–2462

Ishitani M, Liu J, Halfter U, Kim CS, Shi W, Zhu JK (2000) SOS3

function in plant salt tolerance requires N-myristoylation and

calcium binding. Plant Cell 12:1667–1678

Jansson S, Douglas CJ (2007) Populus, a model system for plant

biology. Annu Rev Plant Biol 58:435–458

Katiyar-Agarwal S, Zhu J, Kim K, Agarwal M, Fu X, Huang A, Zhu

JK (2006) The plasma membrane Na?/H? antiporter SOS1

interacts with RCD1 and functions in oxidative stress tolerance

in Arabidopsis. Proc Natl Acad Sci USA 103:18816–18821

Kinclova O, Ramos J, Potier S, Sychrova H (2001) Functional study

of the Saccharomyces cerevisiae Nha1p C-terminus. Mol

Microbiol 40:656–668

Laurie S, Feeney KA, Maathuis FJM, Heard PJ, Brown SJ, Leigh RA

(2002) A role for HKT1 in sodium uptake by wheat roots. Plant J

32:139–149

Lin H, Yang Y, Quan R, Mendoza I, Wu Y, Du W, Zhao S,

Schumaker KS, Pardo JM, Guo Y (2009) Phosphorylation of

SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 protein

kinase stabilizes their protein complex and regulates salt

tolerance in Arabidopsis. Plant Cell 21:1607–1619

Liu J, Zhu JK (1998) A calcium sensor homolog required for plant

salt tolerance. Science 280:1943–1945

Liu J, Ishitani M, Halfter U, Kim CS, Zhu JK (2000) The Arabidopsisthaliana SOS2 gene encodes a protein kinase that is required for

salt tolerance. Proc Natl Acad Sci USA 97:3703–3734

Luo ZB, Polle A (2009) Wood composition and energy content in a

poplar short rotation plantation on fertilized agricultural land in a

future CO2 atmosphere. Glob Change Biol 15:38–47

Martin L, Leblanc-Fournier N, Azri W, Lenne C, Henry C, Coutand

C, Julien JL (2009) Characterization and expression analysis

under bending and other abiotic factors of PtaZFP2, a poplar

gene encoding a Cys2/His2 zinc finger protein. Tree Physiol 29:

125–136

Martinez-Atienza J, Jiang X, Garciadeblas B, Mendoza I, Zhu JK,

Pardo JM, Quintero FJ (2007) Conservation of the salt overly

sensitive pathway in rice. Plant Physiol 143:1001–1012

Mumberg D, Muller R, Funk M (1995) Yeast vectors for the

controlled expression of heterologous proteins in different

genetic backgrounds. Gene 156:119–122

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bioassays with tobacco tissue cultures. Physiol Plant 15:473–495

Niu X, Bressan RA, Hasegawa PM, Pardo JM (1995) Ion homeostasis

in NaCl stress environments. Plant Physiol 109:735–742

Oh DH, Leidi E, Zhang Q, Hwang SM, Li Y, Quintero FJ, Jiang X,

D’Urzo MP, Lee SY, Zhao Y, Bahk JD, Bressan RA, Yun DJ,

Pardo JM, Bohnert HJ (2009) Loss of halophytism by interfer-

ence with SOS1 expression. Plant Physiol 151:210–222

Oh DH, Lee SY, Bressan RA, Yun DJ, Bohnert HJ (2010)

Intracellular consequences of SOS1 deficiency during salt stress.

J Exp Bot 61:1205–1213

Olıas R, Eljakaoui Z, Li J, De Morales PA, Marın-Manzano MC,

Pardo JM, Belver A (2009) The plasma membrane Na?/H?

antiporter SOS1 is essential for salt tolerance in tomato and

affects the partitioning of Na? between plant organs. Plant Cell

Environ 32:904–916

Ottow EA, Polle A, Brosche M, Kangasjarvi J, Dibrov P, Zorb C,

Teichmann T (2005) Molecular characterization of PeNhaD1,

the first member of the NhaD Na?/H? antiporter family of plant

origin. Plant Mol Biol 58:75–88

Parida A, DAS AB, Mittra B (2004) Effects of salt on growth, ion

accumulation, photosynthesis and leaf anatomy of the mangrove,

Bruguiera parviflora. Trees Struct Funct 108:167–174

Pilate G, Guiney E, Holt K, Petit-Conil M, Lapierre C, Leple JC,

Pollet B, Mila I, Webster EA, Marstorp HG, Hopkins DW,

Jouanin L, Boerjan W, Schuch W, Cornu D, Halpin C (2002)

Field and pulping performances of transgenic trees with altered

lignification. Nat Biotechnol 20:607–612

Qiu QS, Guo Y, Dietrich MA, Schumaker KS, Zhu JK (2002)

Regulation of SOS1, a plasma membrane Na?/H? exchanger in

Arabidopsis thaliana, by SOS2 and SOS3. Proc Natl Acad Sci

USA 99:8436–8441

Plant Mol Biol (2010) 74:367–380 379

123

Qiu QS, Barkla BJ, Vera-Estrella R, Zhu JK, Schumaker KS (2003)

Na?/H? exchange activity in the plasma membrane of Arabid-opsis. Plant Physiol 132:1041–1052

Quan R, Lin H, Mendoza I, Zhang Y, Cao W, Yang Y, Shang M,

Chen S, Pardo JM, Guo Y (2007) SCaBP8/CBL10, a putative

calcium sensor, interacts with the protein kinase SOS2 to protect

Arabidopsis shoots from salt stress. Plant Cell 19:1415–1431

Quintero FJ, Ohta M, Shi H, Zhu JK, Pardo JM (2002) Reconstitution

in yeast of the Arabidopsis SOS signaling pathway for Na?

homeostasis. Proc Natl Acad Sci USA 99:9061–9066

Rubio F, Gassmann W, Schroeder JI (1995) Sodium driven potassium

uptake by the plant potassium transporter HKT1 and mutations

conferring salt tolerance. Science 270:1660–1663

Sheen J (2001) Signal transduction in maize and Arabidopsismesophyll protoplasts. Plant Physiol 127:1466–1475

Shi H, Ishitani M, Kim C, Zhu JK (2000) The Arabidopsis thalianasalt tolerance gene SOS1 encodes a putative Na?/H? antiporter.

Proc Natl Acad Sci USA 97:6896–6901

Shi H, Quintero FJ, Pardo JM, Zhu JK (2002) The putative plasma

membrane Na?/H? antiporter SOS1 controls long-distance Na?

transport in plants. Plant Cell 14:465–477

Shi H, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of a plasma

membrane Na?/H? antiporter gene improves salt tolerance in

Arabidopsis thaliana. Nat Biotechnol 21:81–85

Singh B (1998) Biomass production and nutrient dynamics in three

clones of Populus deltoides planted on Indogangetic plains. Plant

Soil 203:15–26

Sun J, Chen S, Dai S, Wang R, Li N, Shen X, Zhou X, Lu C, Zheng X,

Hu Z, Zhang Z, Song J, Xu Y (2009) NaCl-induced alternations

of cellular and tissue ion fluxes in roots of salt-resistant and salt-

sensitive poplar species. Plant Physiol 149:1141–1153

Sun J, Wang MJ, Ding MQ, Deng SR, Liu MQ, Lu CF, Zhou XY,

Shen X, Zheng XJ, Zhang ZK, Song J, Hu ZM, Xu Y, Chen SL

(2010) H2O2 and cytosolic Ca2? signals triggered by the PM

H?-coupled transport system mediate K?/Na? homeostasis in

NaCl-stressed Populus euphratica cells. Plant Cell Environ

33:943–958

Tuskan GA et al (2006) The genome of black cottonwood, Populustrichocarpa (Torr., Gray). Science 313:1596–1604

Volkov V, Wang B, Dominy PJ, Fricke W, Amtmann A (2004)

Thellungiella halophila, a salt-tolerant relative of Arabidopsisthaliana, possesses effective mechanisms to discriminate

between potassium and sodium. Plant Cell Environ 27:1–14

Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C,

Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K,

Kudla J (2004) Visualization of protein interactions in living

plant cells using bimolecular fluorescence complementation.

Plant J 40:428–438

Wang JY, Xia XL, Wang JP, Yin WL (2008) Stress responsive zinc-

finger protein gene of Populus euphratica in tobacco enhances

salt tolerance. J Integr Plant Biol 50:56–61

Wu Y, Ding N, Zhao X, Zhao M, Chang Z, Liu J, Zhang L (2007)

Molecular characterization of PeSOS1, the putative Na?/H?

antiporter of Populus euphratica. Plant Mol Biol 65:1–11

Xiong LM, Schumaker KS, Zhu JK (2002) Cell signaling during cold,

drought and salt stress. Plant Cell 14 suppl:S165–S183

Xu H, Jiang X, Zhan K, Cheng X, Chen X, Pardo JM, Cui D (2008)

Functional characterization of a wheat plasma membrane Na?/

H? antiporter in yeast. Arch Biochem Biophys 473:8–15

Yang Q, Chen ZZ, Zhou XF, Yin HB, Li X, Xin XF, Hong XH, Zhu

JK, Gong Z (2009) Overexpression of SOS (salt overly sensitive)

genes increases salt tolerance in transgenic Arabidopsis. Mol

Plant 2:22–31

Ye CY, Zhang HC, Chen JH, Xia XL, Yin WL (2009) Molecular

characterization of putative vacuolar NHX-type Na?/H?

exchanger genes from the salt-resistant tree Populus euphratica.

Physiol Plant 137:166–174

Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts:

a versatile cell system for transient gene expression analysis. Nat

Protoc 2:1565–1572

Zhang F, Wang Y, Yang Y, Wu H, Wang D, Liu J (2007)

Involvement of hydrogen peroxide and nitric oxide in salt

tolerance resistance in the calluses from Populus euphratica.

Plant Cell Environ 30:775–785

Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71

Zhu JK (2002) Salt and drought stress signal transduction in plants.

Annu Rev Plant Biol 53:247–273

Zhu JK (2003) Regulation of ion homeostasis under salt stress. Curr

Opin Plant Biol 6:441–445

380 Plant Mol Biol (2010) 74:367–380

123