Molecular Cloning and Characterization of a Plant Serine Acetyltransferase Playing a Regulatory Role in Cysteine Biosynthesis from Watermelon

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REGULAR PAPER

Molecular cloning and characterization of plasma membrane-and vacuolar-type Na+/H+ antiporters of an alkaline-salt-tolerantmonocot, Puccinellia tenuiflora

Shio Kobayashi • Natsuki Abe • Kaoru T. Yoshida •

Shenkui Liu • Tetsuo Takano

Received: 31 October 2011 / Accepted: 26 December 2011

� The Botanical Society of Japan and Springer 2012

Abstract A better understanding of salt tolerance in

plants might lead to the genetic engineering of crops that

can grow in saline soils. Here we cloned and characterized

plasma membrane and vacuolar Na?/H? antiporters of a

monocotyledonous alkaline-tolerant halophyte, Puccinellia

tenuiflora. The predicted amino acid sequence of the

transporters were very similar to those of orthologs in

monocotyledonous crops. Expression analysis showed that

(1) NHA was more strongly induced by NaCl in the roots of

P. tenuiflora while in rice it was rather induced in the

shoots, suggesting that the role of NHA in salt excretion

from the roots partly accounts for the difference in the

tolerance of the two species, and that (2) NHXs were spe-

cifically induced by NaHCO3 but not by NaCl in the roots

of both species, suggesting that vacuolar-type Na?/H?

antiporters play roles in pH regulation under alkaline salt

conditions. Overexpression of the antiporters resulted in

increased tolerance of shoots to NaCl and roots to

NaHCO3. Overexpression lines exhibited a lower Na?

content and a higher K? content in shoots under NaCl

treatments, leading to a higher K?/Na? ratio.

Keywords Na?/H? antiporter � Puccinellia tenuiflora �Saline-alkali stress � Salt stress

Introduction

Soil salinity is one of the major environmental stresses

faced by crops today. More than 6% of the world’s land

area ([800 million hectares) is adversely affected by salt

(Munns and Tester 2008). In Northeast China, about

3.2 9 106 ha contain elevated levels of alkaline salt. In this

area, soil pH is more than 9.8, and the characteristics of the

soil are classified as ‘‘alkali’’ or ‘‘saline-alkali’’ (Wang

et al. 2009b). Plants in this area suffer not only from

salinity stress, but also from high pH. The former is mainly

caused by Na?, and the latter is an effect of HCO3- and

CO32-. These properties make it impossible for normal

crops to grow, and salt-tolerant pasture plants and weeds

are dominant in the area. One of these plants is a mono-

cotyledonous graminaceous halophyte, Puccinellia tenu-

iflora (Griseb.) Scrib. et Merr., called alkali grass in

Chinese (Peng et al. 2004). Under saline conditions, P.

tenuiflora exhibits a high K?/Na? ratio, resulting from the

high K?/Na? selectivity of its plasma membrane (Peng

et al. 2004) and a high ability to limit Na? influx in the

roots (Wang et al. 2009a). In addition, the leaves secrete

salts with wax from the stomata (Guorong et al. 2005). EST

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10265-012-0475-9) contains supplementarymaterial, which is available to authorized users.

S. Kobayashi � N. Abe � T. Takano (&)

Asian Natural Environmental Science Center (ANESC),

The University of Tokyo, 1-1-1 Midori-cho,

Nishitokyo, Tokyo 188-0002, Japan

e-mail: [email protected]

S. Kobayashi

e-mail: [email protected]

N. Abe

e-mail: [email protected]

K. T. Yoshida

Department of Ecosystem Studies, The University of Tokyo,

1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

e-mail: [email protected]

S. Liu

Alkali Soil Natural Environmental Science Center,

Northeast Forestry University, Harbin 150040, China

e-mail: [email protected]

123

J Plant Res

DOI 10.1007/s10265-012-0475-9

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and microarray analyses of NaHCO3-stressed plants

revealed two major groups of genes whose expression is

changed by the treatment, suggesting that P. tenuiflora has

at least two stress tolerance pathways (Wang et al. 2007a,

b, c). Several P. tenuiflora genes have been cloned and

characterized and have been implicated in stress tolerance

(Ardie et al. 2009, 2010, 2011; Liu et al. 2009; Wang et al.

2011). Since P. tenuiflora is a monocot, an understanding

of its tolerance mechanisms may help to engineer salt

tolerant cereal crops.

Na? adversely affects plant growth in many ways.

Overaccumulation of Na? in the cytosol may lead to a

reduced K?/Na? ratio, dissipation of the membrane

potential, inhibition of enzyme activities, and production of

reactive oxygen species (Tuteja 2007). Plants avoid Na?

accumulation in the cells mainly by three mechanisms: (1)

restriction of Na? entry into the roots, (2) sequestration of

Na? into the vacuole, and (3) pumping Na? that entered

the cytosol back to the growth medium or to apoplastic

spaces (Apse and Blumwald 2007). Cation pumps, carriers

and channels participate in these processes. Sodium/proton

exchangers, which are a group of electroneutral monova-

lent cation/proton antiporters (CPAs) belonging to the

CPA1 family, play important roles in Na? extrusion and

sequestration (Rodrıguez-Rosales et al. 2009).

NHA (Na?/H? antiporter) or SOS1 (salt overly sensitive

1) is a plasma membrane-type antiporter, and participates in

both Na? extrusion at the roots and Na? loading into the

xylem (Shi et al. 2000, 2002). A plant NHA was first cloned

and characterized in a salt-sensitive Arabidopsis mutant, salt

overly sensitive 1. The gene (AtSOS1) was shown to be

responsible for the salt-sensitive phenotype (Shi et al. 2000;

Wu et al. 1996). The Arabidopsis overexpression line of

AtSOS1 was tolerant to Na? stress (Shi et al. 2003).

Orthologous genes were cloned from crops such as rice,

tomato, and quinoa (Martınez-Atienza et al. 2007; Maughan

et al. 2009; Olıas et al. 2009) and also from halophytes such

as Populus euphratica and reed grass, and more recently

from P. tenuiflora (Takahashi et al. 2009; Wang et al. 2011;

Wu et al. 2007).

The NHX (Na?/H? exchanger) is a vacuolar-type anti-

porter. In plants, it was first reported in Arabidopsis and

designated AtNHX1 (Apse 1999). Whereas plants usually

have only one copy of a plasma-membrane type NHA,

NHXs are normally found as multiple copies: Arabidopsis

has 6 copies (AtNHX1-6) and rice has 5. AtNHX1 and its

orthologs are the best characterized. The atnhx1 mutant

was sensitive to salt stress, and the overexpression line was

tolerant to salt stress (Apse 1999; Apse et al. 2003).

In this study, we focused on the Na?/H? antiporters

NHA and NHX1 in P. tenuiflora, hypothesizing that they

account for the high salt tolerance of the plant. Wang et al.

(2011) cloned a NHA of P. tenuiflora (PtNHA1) and

revealed that it was induced by NaCl, and that Arabidopsis

plants overexpressing PtNHA1 were more tolerant to NaCl

and had higher K?/Na? ratio under NaCl treatment than

WT. We cloned PtNHA1 independently, and also cloned a

cDNA which showed high sequence similarity with rice

NHX1, and named it PutNHX. We used rice homologs for

comparison, since rice, like P. tenuiflora, is a monocot. We

used not only NaCl but also alkaline salts to characterize

the Na?/H? antiporters, since P. tenuiflora grows in a

highly alkaline condition in the wild. The roles of PtNHA1

and PutNHX in neutral or alkaline salt tolerance of plants

were examined using rice overexpression lines.

Materials and methods

Plant materials, growth conditions, and stress

treatments

Seeds of P. tenuiflora were collected in an alkaline soil area

located in North-East China. Seeds were germinated in tap

water for 2 weeks and then transferred to nutrient solution.

Seeds of Oryza sativa L. cv. Nipponbare were harvested at

Institute for Sustainable Agro-ecosystem services, Univer-

sity of Tokyo, in Tokyo, Japan. The seeds were washed with

tap water and germinated in 2.5% PPMTM (Plant Cell

Technology, USA), and subsequently grown in nutrient

solution. Nutrient solution contained 6 mg/l (NH4)2SO4,

2 mg/l K2SO4, 8.2 mg/l MgSO4, 2.3 mg/l KNO3, 7.5 mg/l

Ca(NO3)2, 3.1 mg/l KH2PO4, 10 mg/l Fe-EDTA and the pH

was adjusted to 5.7 with 1 M KOH. Growth chamber was

maintained at 28�C during the day and 22�C at night while

the daily photoperiod of 350–400 lmol m-2 s-1 was 12 h.

NaCl or NaHCO3 were added to the nutrient solution for

stress treatment.

To compare salt stress tolerance, 2-week-old plants of P.

tenuiflora and 1-week-old rice plants were transferred to

nutrient solution containing 0, 100, 300, or 1,000 mM

NaCl or 0, 100, 300, 1,000 mM NaHCO3 and grown for

another 5 days.

Cloning of PtNHA1 and PutNHX

Total RNA was extracted using ISOGEN� (NIPPON

GENE, Japan) from the shoots and roots of P. tenuiflora

plants which were sown 10 days before sampling, accord-

ing to the manufacturer’s instructions. First strand cDNA

was synthesized from the total RNA using Oligo dT primer

and PrimeScript� Reverse Transcriptase (TaKaRa, Japan).

The partial cDNA fragments were amplified by PCR, using

primers designed based on the conserved regions of NHA

and NHX (Table S1) and the first strand cDNA as a

template. Full-length cDNA was obtained with 50- and

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30-RACE techniques. 50-Full RACE CORE Set (TaKaRa,

Japan) and Cap FishingTM 30-Full-Length cDNA Premix Kit

(Seegene, Korea) were used for 50- and 30-RACE, respec-

tively. The PCR product was subcloned and sequenced.

Primers used for full-length amplification and subcloning into

Gateway� vectors (Invitrogen, USA) are shown in Table S1.

Hydropathy analyses and domain analyses were performed

using the SOSUI program (http://bp.nuap.nagoya-u.ac.

jp/sosui/) and Pfam sequence search (http://pfam.sanger.

ac.uk/), respectively.

Real-time RT-PCR

For real-time RT-PCR, total RNA was extracted by the

guanidium isothiocyanate (GTC) method from roots and

shoots of 4-week-old P. tenuiflora or 2-week-old rice

subjected to 3 days of 300 mM NaCl or 300 mM NaHCO3

treatment. DNase-treated RNA was reverse transcribed

to cDNA using High Capacity RNA-to-cDNATM Kit

(Applied Biosystems, USA) following the manufacturer’s

instructions. The cDNA was diluted 10 times and 1 ll of

the diluted cDNA was used as the template for quantitative

RT-PCR analysis. FastStart Universal SYBR Green Master

(ROX) (Roche, Switzerland) and StepOneTM Real-Time

PCR System (Applied Biosystems, USA) were used for

cDNA amplification. A tubulin gene from P. tenuiflora

(PutTubulin) was cloned and used as an internal standard

to normalize the expression data. Primers used for the

amplification of PtNHA1, PutNHX and PutTubulin are

shown in Table S2. 25S rRNA primers introduced by Jain

et al. (2006) were used for an internal standard of rice.

Primers used for the amplification of OsSOS1 and OsNHX1

is also shown in Table S2. The PCR was performed as

follows: 95�C for 10 min, followed by 40 cycles of 95�C

for 15 s and 60�C for 30 s. The experiments were carried

out in triplicate. A ten-fold serial dilution of 1/5 cDNA

mixture of all treatments were used for the standard curve.

Generation of transgenic rice plants overexpressing

PtNHA1 and PutNHX genes

PtNHA1 and PutNHX were subcloned into pActnos/Hm2

vector. The vector supports the Gateway� system (Invit-

rogen, USA), and carries a hygromycin resistance gene.

PtNHA1 and PutNHX were under the control of the actin

promoter. The resultant pActnos-PtNHA1 and pActnos-

PutNHX constructs were transformed into rice plants (cv.

Nipponbare) by Agrobacterium (strain EHA 105)-mediated

transformation of calli (Toki 1997). Transformants selected

on plates containing hygromycin were transferred to soil

and the T0 seeds were obtained by self-pollination.

Hygromycin-based selection and plant cultivation were

repeated to obtain the T1 and T2 seeds. Total RNA was

extracted from 3-week-old T2 plants using RNeasy� Plant

Mini Kit (QIAGEN, Germany) and the expression levels of

PtNHA1 or PutNHX mRNA were checked by RNA gel

blotting (Fig. S1). Probes were designed based on the

30-UTR sequences of PtNHA1 and PutNHX, and the

primers used are presented in Table S3. The T2 plants were

used for stress tolerance tests.

Characterization of transgenic rice plants

overexpressing PtNHA1 or PutNHX

T2 seeds were sterilized and grown on nutrient solution in a

growth chamber for 2 weeks before stress treatments.

To compare stress tolerance, plants were subjected to

150 mM NaCl and 60 mM NaHCO3 for 3 days, and sub-

sequently transferred to nutrient solution without NaCl/

NaHCO3, and allowed to grow for another 1 week. For the

measurement of shoot and root lengths, plants subjected to

100 mM NaCl or 50 mM NaHCO3 for 3 days were used.

To measure ion contents, 14-day-old plants were treated

with 100 mM NaCl or 50 mM NaHCO3 for 3 days. Roots

were separated from shoots and washed thoroughly by tap

water. The roots and shoots were dried in an oven at 70�C

for 3 days. Dried plants were crushed with Multi-beads

shocker� (Yasui kikai, Japan) and inubated in 1 M HCl

overnight, gently shaking. Ion concentrations were mea-

sured using an atomic absorption photometer AA-670G

(Shimadzu, Japan). The experiment was performed in

triplicate.

Results

Growth of P. tenuiflora and rice under salt stress

conditions

Treatment with 100 mM NaCl significantly inhibited

the growth of rice but slightly increased the growth of

P. tenuiflora (Fig. 1). The growth of P. tenuiflora was not

significantly inhibited by NaHCO3 at concentrations up to

300 mM. On the other hand, 100 mM NaHCO3 had det-

rimental effect on the growth of rice.

Cloning and characterization of PtNHA1 and PutNHX

We cloned NHA and NHX from P. tenuiflora. PtNHA1 has

already been cloned by Wang et al. (2011), but we inde-

pendently cloned it using our original primer pair shown in

Table S1. Primers designed to amplify the conserved

region of the NHA and NHX genes in P. tenuiflora were

based on existing plant NHA and NHX genes. They yielded

DNA fragments of 450 and 750 bp, respectively, and their

sequences confirmed that they were partial sequences of

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NHA and NHX. The full-length cDNAs were amplified by

50- and 30-RACE techniques. Since the obtained cDNA

sequence of NHA was a little different from reported

PtNHA1 sequence (the deduced amino acid sequences are

compared in Fig. S2a), we registered it at GenBank

(accession no. AB628205). However, as it has been already

shown that NHA1 of P. tenuiflora is encoded by a single

copy gene (Wang et al. 2011), ours seems to be encoded by

the same gene, PtNHA1. Therefore, we henceforth call it

PtNHA1.

The NHX was named PutNHX (accession no. AB628206).

PutNHX cDNA contained a predicted ORF of 1,617 bp that

appears to encode a protein of 538 amino acids. The protein

is predicted to have 9 transmembrane domains, and a Na?/

H? exchange domain. A putative amiloride binding domain

that is conserved among plant NHXs (Yamaguchi et al.

2003) and a potential glycosylation site, which has also been

predicted for other plant NHXs (Sato and Sakaguchi 2005),

were also conserved in PutNHX (Fig. S2b). In a phylogenic

tree (Fig. S3), PutNHX clustered in the intracellular group

class I, suggesting that it is predicted to be localized at the

vacuolar membrane. Its sequence was highly similar to those

of rice and wheat homologs.

Expression of PtNHA1 and PutNHX compared

with OsSOS1 and OsNHX1

The effect of 300 mM Na? on the expression levels of

NHA was greater in roots than in shoots in both species

(Fig. 2a, b). In rice shoots, NHA expression was increased

more by NaCl treatment than by NaHCO3 treatment, and

peaked after 24 h (Fig. 2b). In P. tenuiflora roots (Fig. 2c),

both treatments increased the PtNHA1 levels dramatically

within the first 12 h. Under NaCl treatment, the level

reached a plateau at 24 h, corresponding to an 11-fold

increase, while under NaHCO3 treatment it peaked at 12 h.

On the other hand, the increase of the expression of NHA in

the rice roots was much smaller under both treatments than

in the P. tenuiflora roots.

The NHX expression level in P. tenuiflora shoots

exposed to NaCl and NaHCO3 peaked after 1 h (Fig. 3a).

Under NaHCO3, it even decreased below the control level

after the peak. On the other hand, in rice shoots (Fig. 3b),

the expression of OsNHX1 increased dramatically with

time under both treatments. One difference was that under

NaCl treatment, the expression peaked at 24 h, whereas

Fig. 1 Growth comparison of rice (left) and P. tenuiflora (right)under salt stresses. Two-week-old P. tenuiflora and 1-week-old

rice plants were subjected to the indicated salt stresses for 5 days.

a, b NaCl-treated rice and P. tenuiflora, respectively. c, d NaHCO3

treatments. Black bars 5 cm

Fig. 2 Effects of NaCl and

NaHCO3 on expression of

NHAs. a, b The relative

expression levels in the shoots

of P. tenuiflora and rice,

respectively, and c, d the values

in the roots of P. tenuiflora and

rice. Tubulin of P. tenuifloraand 25SrRNA of rice were used

as internal controls,

respectively, and the values are

shown as the relative expression

compared to the internal

controls. The relative expression

level in untreated plants (0 h)

was defined as 1. Shown are

mean ± SD (n = 3)

J Plant Res

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under NaHCO3 treatment, it was still increasing at 48 h.

The expression levels of NHXs in roots were drastically

higher under NaHCO3 treatment than under NaCl treatment

in both species, suggesting that this response is specific to

an alkaline salt. The increase in expression started earlier in

rice than in P. tenuiflora.

Overexpression of PtNHA1 and PutNHX in rice

PtNHA1 or PutNHX-overexpressing rice plants were gen-

erated. The mRNA expression was confirmed by RNA gel

blotting (Fig. S1). Three days of 150 mM NaCl treatment

severely damaged the WT plant, but only moderately

affected the shoots of PtNHA1-OX3 and PutNHX-OX3

plants, both of which produced new leaves (Fig. 4a–c). On

the other hand, 3 days of 60 mM NaHCO3 treatment had a

detrimental effect on the shoots of the plants (Fig. 4d–f).

After 1 week of recovery, the WT and PtNHA1-OX3

plants produced only a few new leaves, whereas the Put-

NHX-OX3 overexpression line died. In some of the over-

expression lines, shoot length was less affected by NaCl

than it was in the WT (Fig. 4g, white bars), whereas under

NaHCO3 stress no significant difference in relative shoot

lengths was observed (Fig. 4g, black bars). On the other

hand, relative root lengths under NaCl treatment were not

significantly different between WT and the overexpression

lines (Fig. 4h, white bars), whereas the roots of NHA OX-6

and NHX OX-8 were more tolerant to NaHCO3 than the

WT (Fig. 4h, black bars).

Ion contents were measured using the overexpression

lines PtNHA1-OX1, PtNHA1-OX3, PtNHA1-OX6, Put-

NHX-OX3 and PutNHX-OX8. Shoots of the transgenic

lines had significantly lower Na? contents after 100 mM

NaCl treatment (Fig. 5a) and higher K? contents after

NaCl and 50 mM NaHCO3 treatments compared to WT

(Fig. 5c), although in NaHCO3-treated shoots and roots,

the differences were not so obvious (Fig. 5a–d). The Na?/

K? ratios in shoots were significantly lower in the over-

expression lines than in the WT under both treatments, but

in roots no significant difference was observed (Fig. 5e, f).

Discussion

We focused on the Na?/H? antiporters NHA and NHX as

candidates for the strong tolerance of P. tenuiflora to salt

stress. First we cloned NHA and NHX from P. tenuiflora.

The primary sequence of the NHA we obtained was a little

different from PtNHA1 (Wang et al. 2011), and also from

PtSOS1 (GenBank accession no. GQ452778.1; the three

sequences are shown in Fig. S2a), although it is predicted

that P. tenuiflora has only one copy of NHA (Wang et al.

2011). The minor differences may be the result of the

plants being collected in different places. The NHX-type

antiporter that we cloned seemed to be a homolog of

AtNHX1, which is the most highly expressed type of NHX

in A. thaliana. In an attempt to determine the copy number

of NHX-type antiporters in P. tenuiflora, we tried Southern

blotting using the full-length cDNA sequence of PutNHX

as a probe. However, we could detect only one band (data

not shown). In other plants, NHXs are found in multiple

copies (Fig. S3), so it is unlikely that PutNHX exists in only

one copy in P. tenuiflora. In other plants, it is known that

NHX paralogs share highly conserved sequences at their

transmembrane domains, but the homologies among their

N- and C-termini are low. This may be the reason why we

could detect only one band.

We checked the expression levels of NHA and NHX in

P. tenuiflora, based on the assumption that the expression

levels of the antiporters might be higher in P. tenuiflora

than in rice, leading to higher efficiency of P. tenuiflora to

prevent accumulation of Na? in the cytosol. This was the

Fig. 3 Effects of NaCl and

NaHCO3 on expression of

NHXs. a P. tenuiflora shoots,

b rice shoots, c P. tenuifloraroots and d rice roots. RNA

could not be extracted from the

rice roots subjected to 24 and

48 h of NaHCO3 treatment. The

values are mean ± SD (n = 3)

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case for T. parvula, a halophytic relative of Arabidopsis,

whose salt tolerance was partly attributed to the higher

expression level of SOS1 (Oh et al. 2010a). Our finding of

strong induction of PtNHA1 in roots under NaCl stress is

consistent with the finding of Wang et al. (2011). Moreover,

our finding that the induction level was higher than in rice

(Fig. 2) suggests that the stronger induction of NHA partly

explains the higher tolerance of P. tenuiflora compared to

rice shown in Fig. 1. Wang et al. (2009a) concluded that P.

tenuiflora tolerates Na? stress by limiting the entrance of

Na? at the root, but our result suggests that the Na? extrusion

mechanism partly accounts for the tolerance. The relatively

low expression level of PutNHX in the shoots indicates that

P. tenuiflora cannot easily isolate Na? in the vacuoles in the

leaf cells. Therefore, if P. tenuiflora has a greater ability to

load Na? into the xylem, and therefore into the leaves, the

salt might be exuded through the stomata, as was demon-

strated by Guorong et al. (2005). On the other hand, the

OsNHX1 expression level in the shoots was high, suggesting

that rice leaf cells can remove the excess Na? in the cytosol

by sequestration. NHXs in roots responded differently to

NaCl and NaHCO3 in both rice and P. tenuiflora, suggesting

that plant NHXs are sensitive to pH. ScNHX1, which is a

Na?/H? antiporter localized at the prevacuolar compartment

of the yeast Saccharomyces cerevisiae, has a role in pH

regulation and can affect endosomal trafficking (Ali et al.

2004; Bowers et al. 2000; Brett et al. 2005). Thus, the higher

induction of NHXs by NaHCO3 treatment than by NaCl

treatment may mean that plant NHXs have a role in pH

homeostasis in plant root cells.

Rice plants overexpressing these genes showed slightly

longer shoots after NaCl stress treatment (Fig. 4a–c, g),

consistent with the result of PtNHA1-overexpressing Ara-

bidopsis plants (Wang et al. 2011). On the other hand,

unlike the root length reported by Wang et al., the root

length of the overexpression lines under NaCl treatment

was not significantly different from that of the WT

(Fig. 4h). Possible explanations of this difference are that

rice has a naturally higher tolerance of NaCl than A. tha-

liana, or that our salt treatment was less stressful than the

one used by Wang et al. Under NaHCO3 treatment, shoots

of PtNHA1-OX3 were almost as sensitive as WT shoots

and the shoots of the PutNHX-OX3 lines were even more

sensitive (Fig. 4d–f). Apparently high pH disturbs the

protective role of Na?/H? antiporters against Na? stress, at

least in shoots. Since the roots of some of the overex-

pression lines showed higher tolerance to NaHCO3, Na?/

H? antiporters may help maintain pH homeostasis in roots.

The shoots of transgenic lines showed lower Na? contents

and higher K? contents compared to WT under NaCl stress

condition (Fig. 5a, c), and showed higher K?/Na? ratios

(Fig. 5e). This result, together with the increased tolerance

of overexpression lines to NaCl stress (Fig. 4), suggests

that PtNHA1 and PutNHX have some roles in the tolerance

of P. tenuiflora to NaCl. Although Wang et al. (2011)

previously reported that overexpression of PtNHA1 in

Arabidopsis plants led to higher K?/Na? ratios in both

shoots and roots, in our experiment, roots of the overex-

pression lines showed no significant difference in K?/Na?

Fig. 4 NaCl stress tolerance of WT and transgenic rice plants.

Two-week-old WT (a, d), PtNHA1-OX3 (b, e) and PutNHX-OX3

(c, f) plants were subjected to 150 mM NaCl (a–c) or 60 mM NaHCO3

stress (d–f) for 3 days, and then transferred to the nutrient solution

without salt and allowed to grow for one more week. g, h shows relative

g shoot and h root length of transgenic lines after treatment with

100 mM NaCl or 50 mM NaHCO3 treatments for 3 days. Values are

expressed relative to the control. Asterisks indicate values that are

significantly different from those in the WT under salt treatment

(P \ 0.05 in Student’s t test). n = 3

J Plant Res

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ratios under salt stress compared to WT (Fig. 5). This dis-

crepancy might be due to the use of different plant materials

or different stress conditions in the two studies. It will be

necessary to use more transgenic plants in the future to

clarify the reason. Under NaHCO3 treatment, many of the

transgenic lines failed to limit the increase of Na? content in

the shoots (Fig. 5a). The finding that the K?/Na? ratios of all

the transgenic lines under NaHCO3 treatment were still

higher than those of WT (Fig. 5e) suggests that PtNHA1 and

PutNHX are also involved in Na? homeostasis under alkaline

salt stress, but their contributions seem to be limited. There is

a possibility that constitutive expression of the transporters

led to an imbalance of pH in the plants, which might have

disturbed the activities of other ion transporters. The plasma

membrane-type Na?/H? antiporter in A. thaliana (AtSOS1)

is known to affect the expression levels of other proteins that

maintain the H? gradient, such as H?-ATPase, resulting in

changes in membrane potential and vacuolar pH (Shabala

et al. 2005; Oh et al. 2010b). Another possibility is that the

activities of PtNHA1 and PutNHX, although induced by

NaHCO3 (Figs. 2, 3), were insufficient to maintain ion

homeostasis under severe alkaline stress. Since Na?/H?

antiporters use an H? gradient for Na? transport, their

activities might decrease at high pH.

The expression level of Na?/H? antiporters in the over-

expression lines shown in Fig. S1 seem to correlate with the

K?/Na? ratio under control conditions (Fig. 5e, f, white bars),

but not under stress conditions (black and gray bars). Also,

relative shoot and root lengths of the rice plants over-

expressing PtNHA1 did not correlate with the expression

level, while those of the plants overexpressing PutNHX seem

to correlate with the expression level (Fig. 4). The salt treat-

ments may have affected the stability of the antiporters or

interrupted their function, and thus have masked the effect of

overexpression. Further analysis are needed to clarify if (1)

NHA and NHX activities are actually affected by high pH, and

(2) NHX contributes to pH homeostasis in the plant cell.

Acknowledgments This work was supported by a Grant-in-aid for

Scientific Research (21380002) to T.T. and by JSPS AA Science

Platform Program.

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Fig. 5 Na? and K? contents in

the roots and shoots of the

overexpression lines.

a Na? contents in the shoots.

b Na? contents in the

roots. c K? contents in the

shoots. d K? contents in

the roots. e K?/Na? ratio in the

shoots. f K?/Na? ratio in the

roots. Asterisks indicate values

that are significantly different

from those in the WT (P \ 0.05

in Student’s t test). n = 3

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