© 2015. Published by The Company of Biologists Ltd.

Arabidopsis AINTEGUMENTA (ANT) Mediates Salt Tolerance

by Trans-repressing SCABP8

Lai-Sheng Meng＊1,3, 4 , Yi-Bo Wang2, Shun-Qiao Yao1 and Aizhong Liu

＊1

1. Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of

Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, PR

China.

2. School of Bioengineering and Biotechnology, Tianshui Normal University, 741001,

PR China.

3. Division of Molecular Life Sciences, Pohang University of Science and

Technology, Hyoja-dong, Pohang, Kyungbuk, 790-784, Republic of Korea.

4. School of Life Sciences, University of Science and Technology of China, Hefei,

Anhui Province, 230027, PR China.

＊Corresponding authors: menglaisheng@mail.kib.ac.cn; liuaizhong@mail.kib.ac.cn

ABSTRACT

The Arabidopsis AINTEGUMENTA (ANT) gene, which encodes an APETALA2

(AP2)-like transcription factor, controls plant organ cell number and organ size

throughout shoot development. ANT is thus a key factor in plant shoot development.

Here, we found that ANT plays an essential role in conferring salt tolerance in

Arabidopsis. ant--Knock Out (KO) mutants presented a salt-tolerant phenotype,

whereas 35S:ANT transgenic plants exhibited more sensitive phenotypes under high

salt stress. Further analysis indicated ANT functioned mainly in the shoot response to

salt toxicity. Target gene analysis revealed that ANT bound to the promoter of

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JCS Advance Online Article. Posted on 8 June 2015

SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8), which encodes a putative

calcium sensor, thereby inhibiting SCABP8 expression. It has been reported that the

salt sensitivity of scabp8 is more prominent in shoot tissues. Genetic experiments

indicated that the mutation of SCABP8 suppresses the ant-KO salt-tolerant phenotype,

implying that ANT functions as a negative transcriptional regulator of SCABP8 upon

salt stress. Together, the above results reveal that ANT is a novel regulator of salt

stress, and that ANT binds to the SCABP8 promoter, mediating salt tolerance.

Key-words: AINTEGUMENTA (ANT), SOS3-LIKE CALCIUM BINDING PROTEIN8

(SCABP8)/CALCINEURIN B-LIKE10, salt stress, trans-repression, shoot, Arabidopsis

.

INTRODUCTION

Soil salinity is a major abiotic stress that decreases plant growth, ranking among the

leading factors limiting agricultural productivity. High concentrations of NaCl can

arrest plant development and result in plant death. Salt stress triggers several

interacting events in plants, including the inhibition of enzymatic activity in metabolic

pathways, decreased uptake of nutrients in roots, decreased carbon use efficiency, and

the denaturation of protein and membrane structures.

The SOS (for Salt Overly Sensitive) pathway is thought to play essential roles in

conferring salt tolerance in Arabidopsis thaliana. SOS3, the first gene derived from

this family to be cloned, was obtained via genetic screening of salt-sensitive

mutants and map-based cloning (Liu and Zhu, 1998). SOS3 shows physical

interaction with and activation of SOS2, a protein kinase (Halfter et al., 2000), thus

forming a SOS2-SOS3 complex that in turn activates SOS1, a plasma membrane

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Na+/H+ antiporter (Shi et al., 2000; Qiu et al., 2002). Although calcium is not

required for SOS2 and SOS3 to interact in this process, calcium is thought to increase

the phosphorylation of the synthesized substrate p3 via the SOS2-SOS3 complex

(Halfter et al., 2000). SCABP8/CBL10 as a putative calcium sensor interacts with the

protein kinase SOS2 to protect Arabidopsis shoots from salt stress by modulating ion

homeostasis (Quan et al., 2007; Kim et al., 2007). While similar functions in

biochemical and cellular tests are fulfilled, SCABP8 and SOS3 must perform distinct

regulatory functions in the salt stress response since, for example, they could not

replace each other in genetic complementation experiments (Quan et al., 2007).

The mutations in SOS1, SOS2, and SOS3 all decrease the Na+/H+ exchange

activity and a constitutively active SOS2 enhances Na+/H+ exchange activity in an

SOS1-dependent and SOS3-independent manner (Qiu et al., 2002). The root and

shoot growth of sos1 and sos2 mutants is more significantly inhibited than that of

sos3 under mild salt stress (Zhu et al., 1998). However, SCABP8/CBL10 mainly

protects Arabidopsis shoots from salt stress (Quan et al., 2007; Kim et al., 2007).

Consistent with their function, the expression of SOS1 and SOS2 is detected in both

root and shoot tissues, whereas the expression of SCABP8 is mainly detected in shoot

tissues (Liu et al., 2000; Shi et al., 2002; Quan et al., 2007; Kim et al., 2007).

AINTEGUMENTA (ANT) which encodes a transcription factor of the

AP2-domain family has been found only in plant systems (Mizukami and Fischer,

2000). Like AP2, ANT contains two AP2 domains homologous with the DNA

binding domain of ethylene response element binding proteins (Mizukami and

Fischer, 2000). ANT as a transcription factor is strongly expressed in many dividing

tissues in the plant, in which it plays central roles in developmental processes such

as shoot and flower meristem maintenance, organ size and polarity, flower

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initiation, ovule development, floral organ identity as well as cell proliferation

(Horstman et al., 2014).

Here, we report that ANT mainly protects Arabidopsis shoots from salt stress

and show that SCABP8 is a downstream target of ANT in the regulation of salt stress

since the mutation of SCABP8 suppresses the ant-KO salt-tolerant phenotype,

implying that SCABP8 plays a predominant role in inhibiting ANT-induced salt

tolerance. Thus, ANT is a novel regulator of salt stress, binding to the SCABP8

promoter in order to mediate salt tolerance.

RESULTS

ANT expression patterns

The Arabidopsis ANT gene encoding an AP2-like transcription factor controls plant

organ cell number and organ size throughout shoot development (Mizukami and

Fischer, 2000). ANT is mainly detected in the tissues of activizing division (including

young leaf blades, leaf veins, stem veins, stamens, pistils, meristem tissue and

vascular cylinder) but not in mature tissue or root tissue (including the mature zone of

roots and mature leaves) (Figures 1A-H; also see Mizukami and Fischer., 2000; Elliot

et al., 1996; Klucher et al., 1996). In the roots, ANT was only expressed in the root

tips and vascular cylinder (Figures 1G and H). Thus, it appears that ANT mainly

functions in shoot tissue; this result is consistent with ANT’s role in controlling plant

organ cell number and organ size throughout shoot development (Mizukami and

Fischer, 2000). In this work, we found that the ant-KO mutant, but not 35S:ANT,

exhibited a tolerant phenotype under high salt stress (Figure 2). To examine whether

the expression of ANT is induced by NaCl treatment, total RNA was extracted from

wild-type plants that had been treated with 75 mM NaCl or 100 mM NaCl for 3 and 6

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h. The transcription of ANT was significantly lower at 3 and 6 h after treatment in

comparison with the control plants (Supplemental Figure 1A).

ant-KO and 35S:ANT show different responses to high sodium salt stress

To further determine whether ANT is involved in the regulation of salt stress

response, ant-KO (SALK-022770) and 35S:ANT were analyzed. RT-PCR indicated

that the 35S:ANT lines over-accumulated ANT transcripts compared with the wild

type, whereas ANT was eliminated in the ant-KO (SALK-022770) lines (see

Supplemental Figure 1B).

We sowed seeds of the ant-KO mutant, 35S:ANT and wild-type (Col-0) lines on

solid MS medium with 100 mM NaCl for 30 days. The salt tolerance of the ant-KO

plants was maintained throughout their development from seedlings to mature plants,

whereas a number of 35S:ANT transgenic plants were dry and dead compared with the

wild type (Col-0) (Supplemental Figure 2A). The ant-KO exhibited longer primary

roots, whereas the 35S:ANT showed shorter primary roots than wild type did

(Supplemental Figures 2A and B). The number of ant-KO lateral roots was enhanced,

whereas the number of 35S:ANT lateral roots was not different relative to that of the

wild type (Supplemental Figures 2A and C). Consequently, whereas the overall root

fresh weight of the ant-KO mutant was only ~1.6 times greater than that of the wild

type (Supplemental Figure 2E), shoot fresh weight in the ant-KO mutant was

enhanced by ~2.9 times over the wild type (Col-0), and its leaves were also larger

(Supplemental Figures 2A and D). However, seedling weight of ant-KO, 35S:ANT

and WT was not different on medium without salt (Supplemental Figure 3)

To further characterize the salt insensitivity of the 35S:ANT and ant-KO mutant,

5-day-old seedlings were transferred to solid MS medium with NaCl (75 mM NaCl

and 100 mM NaCl) for 15 days. On solid MS medium with both 75 mM NaCl and

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100 mM NaCl, the length of the ant-KO primary roots was increased, whereas the

length of the 35S:ANT primary roots was not different relative to that of the wild type

(Figures 2A and B). On MS medium with 75 mM NaCl, the number of ant-KO lateral

roots was enhanced, whereas the number of 35S:ANT lateral roots was not

significantly different relative to that of the wild type (Figures 2A and C). However,

on MS medium with 100 mM NaCl, the number of ant-KO lateral roots was

enhanced, while the number of 35S:ANT lateral roots was reduced relative to that of

the wild type (Figures 2A and C). Consequently, whereas the overall root fresh weight

in the ant-KO mutant was increased over that of the wild type (Col-0) by only ~1.16

and ~1.22 times on MS medium with 75 mM NaCl and 100 mM NaCl, respectively

(Figures 2A and E), shoot fresh weight in the ant-KO mutant was ~1.44 and ~ 1.53

times greater on MS medium with 75 mM NaCl and 100 mM NaCl, respectively

(Figures 2A and D). Moreover, shoot fresh weight in the 35S:ANT plants was

decreased on MS medium with 100 mM NaCl ( Figures 2A and D). However,

seedling weight of ant-KO, 35S:ANT and wild type was not different on medium

without salt (Supplemental Figure 3).

To confirm that ANT is involved in regulation of salt stress, we assay whether

ant-KO +35SANT lines (transforming the mutant with a genomic DNA of ANT) can

restore ant-KO less sensitive to similar to wild-type. Five-day-old ant-KO, ant-KO+

35S:ANT, 35S:ANT and Col-0 seedlings were transferred to solid MS medium with

100 mM NaCl for 10 days. On solid MS medium without NaCl, the weight of the

15-day-old shoots was not different (Supplemental Figures 3A and B). However, on

MS medium with 100 mM NaCl, the weight of 15-day-old ant-KO shoots was

enhanced, whereas that of corresponding 35S:ANT was declined (Supplemental

Figures 3A and B). And the weight of shoots between ant-KO+ 35S:ANT and Col-0

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seedlings was not different (Supplemental Figures 3A and B), indicating the ant-KO

mutant phenotype can be rescued.

Together, our findings indicates that ANT functions mainly in the shoot response

to salt toxicity.

ant-KO mutant and 35S:ANT seedlings are not specific to the sodium response

To determine whether the growth defects of the ant-KO mutant and 35S:ANT

seedlings are specific to the sodium response; these seedlings were treated with a few

kinds of salts. With treatment with 100 mM KCl, ant-KO mutant and 35S:ANT

seedlings presented less sensitive and more sensitive relative to wild-type,

respectively (Figures 3A-D). Similarly, with treatment with 10 mM LiCl, ant-KO

mutant and 35S:ANT seedlings also presented less sensitive and more sensitive

relative to wild-type, respectively (Figures 3A-D). Furthermore, with treatment with

combination of both 100 mM KCl and 10 mM LiCl, ant-KO mutant and 35S:ANT

seedlings presented less sensitive and more sensitive relative to single 100 mM KCl

or 10 mM LiCl, respectively (Supplenental Figure 4). However, seedling weight of

ant-KO, 35S:ANT and WT was not different on medium without salt (Supplemental

Figure 4).

ANT negatively modulates SCABP8 at the transcriptional level

To detect the downstream target genes of ANT, the expression levels of the genes

involved in salt stress response were analyzed through quantitative RT-PCR. The

SOS pathway plays an essential role in the salt tolerance of Arabidopsis thaliana.

Thus, SOS1, SOS2, SOS3 and SCABP8 were the first genes examined. Transcripts of

SCABP8 were much more abundant in ant-KO, whereas transcripts of SCABP8 were

lower in 35S:ANT compared with the wild type (Figure 4A). We also observed that

the expression levels of SOS1, SOS2 and SOS3 were not significantly different

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between ant-KO, 35S:ANT and wild-type plants (Figure 4A). Moreover, ANT

transcript abundance was similar between wild-type, sos1, sos2, sos3 and scabp8

plants (Figure 4B). Therefore, ANT negatively modulates SCABP8 expression at the

transcript level. Plant NHX1, NHX4, NHX5 and NHX6 transporters are involved in

salt tolerance (Hernandez et al., 2009; Bassil et al., 2011). These findings

demonstrated that the expression level of these genes was not altered in 35S:ANT

seedlings relative to wild-type (Figure 4G), indicating ANT looks at SCABP8

regulating specifically.

ANT associates with the SCABP8 promoter both In vitro and In vivo

To investigate binding of ANT to SCABP8 promoter, we performed nucleotide

sequence analysis and found that the two AP2 domains in ANT were selectively

associated with the consensus sequence gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t)

(Nole-Wilson and Krizek, 2000; Krizek, 2003). Our nucleotide sequence analysis also

found that the promoters of the SCABP8 gene contained conserved sequence motifs

(Figure 5 and Figure 4C). This portion of ANT corresponds to the two AP2 repeats

and the linker region between them and will be referred to as ANT-AP2R1R2

(Nole-Wilson and Krizek, 2000). The GST fused with the DNA-binding domain in

the ANT-AP2R1R2 was expressed in Escherichia coli, and then these fused proteins

were purified with the help of the GST tag. A gel mobility shift assay was performed

to assay whether the recombinant ANT-AP2R1R2 protein could associate to the

SCABP8 promoter. When the ANT-AP2R1R2 protein and the probes of the

ANT-binding consensus sequences were added, a shifted DNA-binding was

detectable; no band was detected when only the probes of the ANT-binding consensus

sequences were added (Figures 5A and B). When unlabeled probes of the

ANT-binding consensus sequences were added to the reaction mixture, the

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DNA-binding was abolished (Figure 5B). However, the GST-ANT protein was

unable to bind the mutated DNA probes (Figures 5C and D). These data indicate that

the ANT-AP2R1R2 DNA-binding site binds to the SCABP8 promoter In vitro.

To further confirm the binding possibility in vivo, we used transgenic shoots

expressing the 35S:ANT:HA construct for a chromatin immunoprecipitation (ChIP)

analysis. The use of the S1 region (-1456 to -1152 bp) of the primer resulted in greater

amounts of PCR product than the use of the S2 (-1152 to -852 bp) or S3 (-348 to -48

bp) regions (Figures 4D and E). These data clearly indicated that ANT bound to the

SCABP8 promoter in vivo, as was required to suppress SCABP8 expression.

ANT acts genetically upstream of SCABP8

To assay whether ANT acted genetically upstream of SCABP8 in regulating the salt

stress response, we performed a double mutant analysis by combining ant-KO with

scabp8 lines that were insensitive and supersensitive under high salt stress,

respectively. We selected the genotypes from the segregating F3 population by PCR

genotyping. The SCABP8 mutation significantly suppressed the insensitive

phenotypes of the ant-KO line under high salt stress (Figure 6). The length of the

ant-KO primary roots was obviously increased, whereas the lengths of the

ant-KOscabp8 and scabp8 primary roots were significantly reduced relative to that of

the wild type on solid MS medium with NaCl (75 mM NaCl and 100 mM NaCl)

(Figures 6A and B). The overall shoot and root fresh weights in the ant-KO mutants

were increased, whereas those in the ant-KOscabp8 and scabp8 mutants were

decreased relative to those of the wild type on MS medium with 75 mM NaCl and

100 mM NaCl, respectively (Figures 6A, C and D). Together, the above results

indicate that ANT acts genetically upstream of SCABP8 in regulating the salt stress

response (Figure 7).

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DISCUSSION

ANT encoding a transcription factor of the APETALA2 family is involved in cell

proliferation and growth control (Elliott et al., 1996; Klucher et al., 1996; Eshed et al.,

1999; Krizek, 1999; Mizukami and Fischer, 2000; Nole-Wilson and Krizek, 2000;

Mizukami, 2001). Here, we showed that ant-KO mutants presented a salt-tolerant

phenotype, whereas transgenic plants harboring the 35S:ANT construct exhibited

more sensitive phenotype under high salt stress. Under normal growth conditions in

the glasshouse, 35S:ANT exhibited more cell number and larger organ size (seeds,

leaves, flowers, large root systems and higher weight) (Mizukami and Fischer, 2000),

at the same time, 35S:ANT plants were hypersensitivity to salt stress. Thus, the

regulator of plant development might be involved in modulating environmental stress.

Arabidopsis AN3, a transcription co-activator, is an important regulator of leaf and

light-induced root development (Kim and Kende, 2004; Horiguchi et al., 2005; Meng,

2015). Very recently, we found that AN3 controls water-use efficiency (WUE) and

drought tolerance through regulating stomatal density and improving root architecture

via the transrepression of YODA (YDA) (Meng and Yao, 2015). The Arabidopsis E3

SUMO ligase SIZ1 regulates both plant growth and drought tolerance, that is, the null

mutant siz1-3 shows smaller organ size due to reduced expression of genes involved

in brassinosteroid biosynthesis and signaling, at the same time, mutant seedlings of

siz1-3 exhibit significantly lower tolerance to drought stress (Catala et al., 2007).

Loss-of-function of PROTEIN S-ACYL TRANSFERASE10 (PAT10) leads to

pleiotropic growth defects, including smaller leaves, dwarfism, and sterility, while

pat10 mutants exhibits hypersensitive to salt stresses (Zhou et al., 2013). NHX1

(K+/H+) and NHX2 are vacuolar proteins, and they control vacuolar pH and K+

homeostasis and salt tolerance (Bassil et al., 2011). Thus, the role of NHX1 and

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NHX2 is regulating K+/H+ exchange, and this exchange modulates cell expansion in

rapidly elongating tissues, for example, filaments and hypocotyls, and plays unique

and specific roles in flower development (Bassil et al., 2011). We speculate that

similar to the role of NHX1 and NHX2, ANT might be involved in regulating either

plant growth or development by SOS system regulation, which SCABP8/CBL10 as a

putative calcium sensor interacts with the protein kinase SOS2 to protect Arabidopsis

shoots from salt stress by modulating ion homeostasis (Quan et al., 2007; Kim et al.,

2007). The connection between plant development and environmental stress could

open up future research to provide further insights into the relative mechanism that

occurs between the development and stress.

On MS medium supplemented with 100 mM NaCl, sos3, 35S:ANT and scabp8

mutants exhibited severe growth suppression in both shoot and root tissues, however

nearly complete growth arrest occurred only in the root of sos3 and the shoot of

35S:ANT and scabp8 plants (Zhu et al., 1998; Quan et al., 2007; Figure 2). At a lower

NaCl concentration (75 mM), the influence of sos3 mutation on salt tolerance was

significantly suppressed to root growth (Zhu et al., 1998), whereas the influence of the

35S:ANT and scabp8 mutation was more inhibited in shoot growth (Quan et al., 2007;

Figure 2). These findings tell us that salt tolerance in planta under extreme situations

are necessary for the functional integration of all tissues but also that SOS3, ANT and

SCABP8 are needed for salt tolerance in a tissue-specific manner. Consistent with

these findings, their respective expression patterns as assayed via promoter-GUS

fusions (Figure 1; also see Quan et al., 2007). SOS3 was only expressed in root

tissues, particularly in root tips, whereas ANT and SCABP8 was preferentially

expressed in shoot tissues. Thus, SCABP8 and ANT mainly protects shoot tissues,

whereas SOS3 protects root tissues from salt stress, suggesting tissue-specific manner

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may be important for plant doing with environmental stress.

While the expression of SCABP8 was up-regulated by salt stress, the expression

of ANT was down-regulated by salt stress, indicating that ANT and SCABP8 differ in

their transcriptional regulation in response to salinity. Functional analyses, as well as

the specific sodium sensitivity of the sos1, sos2, sos3, and scabp8 mutants, imply that

SOS3 and SCABP8 have overlapping functions in the activation of SOS2 and its

downstream target, SOS1 (Figure 7; also see Quan et al., 2007). Thus, we conclude

that ANT directly binds to SCABP8 promoter for regulating the SOS signaling

cascade in the salt stress response.

MATERIALS AND METHODS

Plant materials and growth conditions

sos1, sos2, sos3 and scabp8 (Quan et al., 2007) mutants in the Col-0 background have

been described previously. ant-KO (SALK-022770), ant-KO+35S:ANT and 35S:ANT

seeds (Ph2GW7; Col-0 background; homozygote; hygromycin) were kindly provided

by Prof. H.G. Nam (DGSIT, Korea). ant-KO (SALK-022770) is a T-DNA mutant, the

single pass sequence of which was recovered from the left border of the T-DNA; and

this sequence lies within 300 bases of the 3' end of At4g37750, as has been described

by ABRC. Transgenic plants were generated using the Agrobacterium

tumefaciens–mediated floral dip method (Zhang et al., 2006). For ant-KO and

35S:ANT identification, the primers F-5'-ATG AAG TCT TTT TGT GAT AAT

GA-3' and R-5'-TTC AAT CTC TTT CTG ATA ATT CTC-3' were used. ant-8

(CS3944) was obtained from ABRC.

Double ant-KOscabp8 mutants were generated through genetic crosses, and

homozygous lines were identified through comparison with the parental phenotype

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and PCR-based genotyping. The ANT gene-specific primers were F-5'-GAG AAG

GAA GAG CAG TGG TTT CT-3' and R-5'-CAG ATG TTC GGA TCC AAT ATG G

-3'; the SCABP8 gene-specific primers have been described previously (Quan et al.,

2007).

Plants exhibiting the 35S:ANT phenotype [large rosette leaves and large seeds

grown in white light (Mizukami and Fischer., 2000)] in the F2 populations were

screened for Dr5:GUS expression in roots. F3 seed was collected from those

exhibiting expression, and lines expressing GUS in all F3 plants were used for

subsequent analysis.

The seeds were subjected to 4°C for 3 days and then sown onto solid Murashige

and Skoog (MS) medium supplemented with 1% sucrose. The roots, seeds, leaves and

embryos were photographed, and then they were determined using Image J software.

The seedlings grown on agar were maintained in a growth room under 16/8 h

light/dark cycles with cool white fluorescent light at 21°C ± 2°C. Plants grown in

soilless media were maintained in a controlled environment growth room under 16/8

h light/dark cycles with cool white fluorescent light at 21°C ± 2°C.

Salt sensitivity

Seeds of the mutant and wild-type (Col-0) plants were sterilized in a solution

containing 30% sodium hypochlorite and 0.1% Triton X-100 for 10 min, washed five

times with sterilized water, and sown on MS medium with 0.8% Phytagel

(Sigma-Aldrich). The plates were placed at 4°C for 3 days, and the seeds were then

germinated vertically at 21°C ± 2°C under continual illumination. Five-day-old

seedlings with a root length of ~1.5 cm were transferred onto MS medium with salt

added as previously described (Quan et al., 2007).

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RT-PCR

Total RNA was extracted from the tissues indicated in the figures using TRIZOL

reagent (Invitrogen), as has been previously described by Yu et al. (2008). SYBR

green was used to monitor the kinetics of the PCR products in the real-time RT-PCR,

as has been previously described by Yu et al. (2008). To analyze the expression levels

of SOS1, SOS2, SOS3 and SCABP8 in Col-0, ant-KO and 35S:ANT seedlings, the

following primers were used: F-5'-ATG ACG ACT GTA ATC GAC GCG AC-3' and

R-5'-CGA ATT CCA TGG CCG ATC TTT C -3' for SOS1; F-5'-ATG ACA AAG

AAA ATG AGA AGA GT -3' and R-5'-CAC AAA CTC CAA AAC TAT ATA T-3'

for SOS2; F-5'- TGC AAT GCG ACC ACC GGG AT-3' and R-5'- TTT CAT GGA

CCG GCG CGC TT-3' for SOS3; F-5'-ATT GCC GCA GCA CAT CGC CT -3' and

R-5'- TGG AGC TTG GAA CAG CGC CAG-3' for SCABP8. To analyze the

expression levels of NHX1, NHX4, NHX5 and NHX6 in Col-0 and 35S:ANT seedlings,

the following primers were used: F-5'-ATG TTG GAT TCT CTA GTG TCG A-3' and

R-5'- ACG AAA TTG CGG AAA AAC TGC TT-3' for NHX1; F-5'-ATG GTG ATC

GGA TTA AGC ACA AT-3' and R-5'-TGG AAG AAG ATA AAT AAA GAA GAG

-3' for NHX2; F-5'-ATG GAG GAA GTG ATG ATT TCT C-3' and R-5'- TTT

AGG TTG AAG ACT GAA ACC TG-3' for NHX5; F-5'-ATG TCG TCG GAG CTG

CAG AT-3' and R-5'-GAA AGA AGA ACT CAT CGT GGA AA -3' for NHX6.

To analyze ANT expression in Col-0, sos1, sos2, sos3 and scabp8 seedlings, the

primers F-5'- AGG TGG CAA GCA CGG ATT GGT -3' and R-5'- AGG CAA CGC

GAA AAT CGC CG-3' were used. To analyze the expression levels of ANT in Col-0,

ant-KO and 35S:ANT seedlings, the following primers were used: F-TCT TCT TCT

GTT CCA CCT CAA C; R-TCT GCA GCT TCT TCT TGG GTT. R-5-AAT ACG

TCG GCG ATT CTC CG-3; F-5-CTT AGG AGA AGG AAA CAC TG-3 were used

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for TUB4.

Plasmid constructs

For the ANT promoter analysis, the promoter-GUS construct At4g37750 was created

by inserting approximately 2.0 Kb promoter fragments into pCB308R, as previously

described by Lei et al. (2007). The primers used for ANT were P1-ggg gac aag ttt gta

caa aaa agc agg ct GTT CAA TAA ATA TGT TTC CAC GTA and P2- ggg gac cac

tttg tac aag aaa gct ggg t CGC TTT TAG CCC TAC AAA TTC C. To obtain the

ANT:HA plasmid (CB2004), we used the primers F-ggg gac aag ttt gta caa aaa agc

agg ct TTC AAT CTC TTT CTG ATA ATT CTC and R--ggg gac cac tttg tac aag aaa

gct ggg t TCA AGA ATC AGC CCA AGC AGC.

GUS staining

Using a buffer mix (1 mM X-gluc, 60 mM NaPO4 buffer, 0.4 mM of

K3Fe(CN)6/K4Fe(CN)6 and 0.1% (v/v) Triton X-100), the samples (transgenic plants

harboring and expressing Pro ANT:GUS ) were stained and then incubated at 37°C for

8 h. After GUS staining, chlorophyll was removed using washes of 30%, 50%, 70%,

90% and 100% ethanol for approximately 30 min each. GUS staining was performed

as previously described by Lei et al. (2007).

ChIP assay

Transgenic lines overexpressing 35S:ANT:HA were used in this assay. ChIP was

performed as previously described (Yoo et al., 2010). HA and GFP tag-specific

monoclonal antibody was used for ChIP analysis. The ChIP DNA products were

analyzed through Q-PCR using primers that were synthesized to amplify ~ 300 bp

DNA fragments in the promoter region of SCABP8. The following primer sequences

were used: S1 'P1-ataaacaattttaatctcgata; P2-attttgcggtatattttgttatt'; S2

'P3-aaaatagacaacaaattctttt; P4-aatcataatatatgacctttga'; S3 'P5-gttgagattccaactagtatat;

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P6- tgtcttatcaaa ctctatagttg'.

Protein expression and purification

The plasmid pGEX-5X-1 for ANT was used in this experiment. The coding sequence

of ANT was amplified using the primer pair (5ʹ-GGA TCC ATG AAG TCT TTT TGT

GA-3ʹ and 5ʹ- GAA TTC AGA ATC AGC CCA AGC-3ʹ ) and cloned into the BamHI

and EcoRI restriction sites of the pET28a plasmid to generate the final plasmid.

Recombinant glutathione S-transferase binding protein (GST)-tagged ANT was

extracted from transformed E. coli (Rosetta2) after 10 h of incubation at 16°C

following induction with 10 µM isopropyl β-D-1-thiogalactopyranoside. These

recombinant proteins were purified using GST-agarose affinity.

Electrophoretic mobility shift assay (EMSA)

The electrophoresis mobility shift assay (EMSA) was performed using the LightShift

Chemiluminescent EMSA Kit (Pierce, 20148) according to the manufacturer’s

instructions. The biotin-labeled DNA fragments (5'-

gaaccgaccagttaaccgataattatacatccggtttgacttacccataatttatccaaaactaatccgattttga-3') and

mutated DNA fragments (5'-gaagggaccagttaagggataattatacatccggttt

gacttacccataatttatccaaaactaatgggattttga-3') were synthesized, annealed and used as

probes, and unlabeled versions of the same DNA fragments were used as competitors

in this assay. The probes were incubated with the ANT-fused protein at room

temperature for 15 min in a binding buffer concentration: 50 mM HEPES-KOH [pH

7.5], 375 mM KCl, 6.25 mM MgCl, 1 mM DTT, 0.5 mg/mL BSA, Glycerol 25%).

Each 20 ml to bind reaction contained 25 fmol biotin-labeled probes and 5 mg

protein, and 1 mg Poly(dIdC) was supplemented to the reaction for minimizing

nonspecific interactions. And then, we analyzed the reaction products via using 8.0 %

native polyacrylamide gel electrophoresis. Electrophoresis was performed at 100 V

for approximately 1-2 h in TGE buffer (containing 12.5 mM Tris, 95 mM glycin, and

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0.5 mM EDTA at pH 8.3, precooled at -12°C). The DNA fragments on the gel were

transferred onto a nitrocellulose membrane with 0.5XTBE at 100 V (~400 mA) for 50

min at 4°C. With cross-linking the transferred DNA to the membrane, the membrane

was incubated in the blocking buffer for 20-30 min under gently shaking, then

transferred to a conjugate/blocking buffer by mixing 33.3 ml stabilized

Streptavidin-Horseradish Peroxidase Conjugate with 10 ml blocking buffer according

to the manufacturer’s protocol. The membrane was washed 4-6 times for 5 min each

with a washing buffer. Biotin-labeled DNA was detected using the chemiluminescent

method in terms of the manufacturer’s protocol.

Acknowledgments

We thank Prof Hong Gil Nam (DGSIT, Korea) and Cheng-Bin Xiang (University of

Science and Technology of China, China) for their support during the initial stage of

this work. ant-KO (SALK-022770) and 35S:ANT [ant-KO (SALK-022770) and

35S:ANT seeds were kindly provided by Prof. H.G. Nam (DGSIT, Korea)]. sos1,

sos2, sos3 and scabp8 seeds were kindly provided by Prof. Y. Guo (Agricultural

University of China, China).

Competing interests

The authors declare no competing interests

Author contributions

L.S. Meng designed experiments. L.S. Meng performed the experiments. L.S. Meng,

S.Q Yao and Y.B Wang completed statistical analysis of data. L.S. Meng and A.Z

Liu wrote, edited and revised this manuscript.

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Funding

We are grateful for National Key Basic Research Program of China (Grant No.

2014CB954100) and National Key Technology R & D Program (2015BAD15B02).

This research was also supported by ‘QingLan’ Talent Engineering Funds by Tianshui

Normal University”.

Supplementary material

Supplementary material available online.

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Figures

Figure 1. ANT Expression Analysis.

Expression of ANT pro:GUS in 4-day-old young leaf blades (A), veins (B), pollen and stamens (C),

stem veins (D), siliques (E), pistils (F), root mature zone (G) and root tips (H).

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Figure 2. ant-KO (KO-knock out)seedlings showed resistance to salt stress.

Seedlings were grown on solid MS medium without NaCl for 5 days and were then transferred to solid

MS medium with 75 mM NaCl or 100 mM NaCl for 15 days.

(A). Representative 20-day-old ant-KO, wild-type (Col-0) and 35S:ANT seedlings grown on solid MS

medium with 75 mM NaCl or 100 mM NaCl for 15 days. Magnification is the same for each image.

(B), (C), (D) and (E). Bar graph exhibiting the differences in primary root length (B), lateral root

number (C), shoot fresh weight (D) and root fresh weight (E) between the 20-day-old ant-KO,

wild-type (Col-0) and 35S:ANT seedlings grown on solid MS medium with 75 mM NaCl or 100 mM

NaCl for 15 days. Error bars represent SD (**P < 0.01, *P < 0.05; n=15).

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Figure 3. ant-KO and 35S:ANT seedlings showed different response from the wild type (Col-0) to KCl

or LiCl.

Seedlings were grown on solid MS medium without NaCl for 5 days and were then transferred to solid

MS medium with 10 mM LiCl or 100 mM KCl for 15 days.

(A). Representative 20-day-old ant-KO (a), wild-type (Col-0) (b) and 35S:ANT (c) seedlings grown on

solid MS medium with 100 mM KCl for 15 days. Representative 20-day-old ant-KO (d), wild-type

(Col-0) (e) and 35S:ANT (f) seedlings grown on solid MS medium with 10 mM LiCl for 15 days.

Magnification is the same for each image. Bars= 2.0cm for (a) to (f).

(B), (C) and (D). Bar graph exhibiting the differences in primary root length (B), lateral root number

(C) and shoot fresh weight (D) between the 20-day-old ant-KO, wild-type (Col-0) and 35S:ANT

seedlings grown on solid MS medium with 100 mM KCl or 10 mM LiCl. Error bars represent SD (*P

< 0.05; n=15).

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Figure 4. ANT negatively regulates SCABP8 at the transcript level.

(A). Bar graph exhibiting the differences of the expression levels of SOS1, SOS2, SOS3 and SCABP8 in

15-day-old ant-KO, wild-type (Col-0) and 35S:ANT seedlings grown under white light conditions.

Error bars represent SD (***P < 0.001,*P < 0.05; n=3). At least five independently propagated lines

were used for each seedling type, and the wild type was set as 1.0. Quantifications were normalized to

the expression of UBQ5. Error bars represent SD (n=3).

(B).Bar graph exhibiting the differences of expression of ANT between wild-type (Col-0), sos1, sos2,

sos3 and scabp8 seedlings grown under white light conditions.

(C). Schematic of the SCABP8 promoter loci and their amplicons for ChIP analysis.

Several amplicons in the SCABP8 promoter were used for ChIP analyses. Additionally, S1 contains

the ANT associating consensus sequences (gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t)). S1, S2 and S3

indicate different three regaions in SCABP8 promoter.

(D) and (E). Chromatin immunoprecipitation (ChIP) analysis. Enrichment of particular SCABP8

chromatin regions with anti-HA antibodies (as a control) or anti-GFP antibodies in 35S:ANT:HA

transgenic shoots (E) and roots (F) as detected by real-time PCR analysis. Quantifications were

normalized to the expression of UBQ5. Error bars represent SD (n=3). Input was set as 100%.

(G). Expression of NHX1, NHX4, NHX5 and NHX6 in Col-0 and 35S:ANT seedlings.

(A). Bar graph exhibiting the differences of the expression levels of NHX1, NHX4, NHX5 and NHX6 in

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15-day-old wild-type (Col-0) and 35S:ANT seedlings grown under white light conditions. Error bars

represent SD (n=3). At least five independently propagated lines were used for each seedling type, and

the wild type was set as 1.0. Quantifications were normalized to the expression of UBQ5. Error bars

represent SD (n=3).

Figure 5. Gel shift analysis

(A) and (C). Gel shift analysis of ANT protein associating to cis-elements in the promoter of SCABP8.

The oligonucleotide sequences of the probe and the mutated form of the probe (mProbe) within the

SCABP8 promoter used in the EMSA. Underlined letters in blue indicate the sequences of the

ANT-recognition motifs. mProbe: ANT-recognition motifs in the probe were mutated as indicated by

big letters.

(B). Unlabeled SCABP8 promoter (2 µg) was used as a competitor to determine the specificity of

DNA-binding activity for ANT.

(D). The mutant version of the SCABP8 promoter (CC/GG) was labeled with biotin and used for

EMSA with ANT polypeptides.

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Figure 6. SCABP8 Mutation Suppresses the ant-KO Mutant Phenotype.

(A). Seedlings were grown on solid MS medium without NaCl for 5 days and were then transferred to

solid MS medium with 75 mM NaCl or 100 mM NaCl for 15 days.

(B). Bar graph exhibiting the differences in primary root length between the indicated 20-day-old

seedlings grown on solid MS medium with 75 mM NaCl or 100 mM NaCl. Error bars represent SD

(**P < 0.01, *P < 0.05; n=12).

(C). Bar graph exhibiting the differences in shoot fresh weight between the indicated 20-day-old

seedlings grown on solid MS medium with 75 mM NaCl or 100 mM NaCl.Error bars represent SD

(**P < 0.01, *P < 0.05; n=12).

(D). Bar graph exhibiting the differences in root fresh weight between the indicated 20-day-old

seedlings grown on solid MS medium with 75 mM NaCl or 100 mM NaCl. Error bars represent SD

(**P < 0.01, *P < 0.05; n=12).

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Figure 7. Salt stress induces downregulation of ANT. ANT associates directly to the SCABP8

promoter and causes the upregulation of SCABP8, thereby inducing salt stress tolerance in the plant. In

vitro, SOS3 physically interacts with and activates an Ser/Thr protein kinase, SOS2. Among the

downstream targets of the SOS3-SOS2 complex is SOS1, a plasma membrane–localized sodium/proton

antiporter (Shi et al., 2000; Qiu et al., 2002; Quintero et al., 2002). SCABP8/CBL10, a putative

calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress

(Quan et al., 2007).

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