RESEARCH ARTICLE

Arabidopsis AINTEGUMENTA mediates salt tolerance bytrans-repressing SCABP8Lai-Sheng Meng1,2,3,*, Yi-Bo Wang1,*, Shun-Qiao Yao2 and Aizhong Liu2,*

ABSTRACTThe Arabidopsis AINTEGUMENTA (ANT) gene, which encodes anAPETALA2 (AP2)-like transcription factor, controls plant organ cellnumber and organ size throughout shoot development. ANT is thusa key factor in the development of plant shoots. Here, we havefound that ANT plays an essential role in conferring salt tolerance inArabidopsis. ant-knockout mutants presented a salt-tolerantphenotype, whereas transgenic plants expressing ANT under the35S promoter (35S:ANT) exhibited more sensitive phenotypes underhigh salt stress. Further analysis indicated that ANT functions mainlyin the shoot response to salt toxicity. Target gene analysis revealedthat ANT bound to the promoter of SOS3-LIKE CALCIUM BINDINGPROTEIN 8 (SCABP8), which encodes a putative Ca2+ sensor,thereby inhibiting expression of SCABP8 (also known as CBL10). Ithas been reported that the salt sensitivity of scabp8 is more prominentin shoot tissues. Genetic experiments indicated that the mutation ofSCABP8 suppresses the ant-knockout salt-tolerant phenotype,implying that ANT functions as a negative transcriptional regulatorof SCABP8 upon salt stress. Taken together, the above results revealthat ANT is a novel regulator of salt stress and that ANT binds to theSCABP8 promoter, mediating salt tolerance.

KEY WORDS: AINTEGUMENTA (ANT), SOS3-LIKE CALCIUMBINDING PROTEIN8, SCABP8, CALCINEURIN B-LIKE10, Salt stress,Trans-repression, Shoot, Arabidopsis

INTRODUCTIONSoil salinity is a major abiotic stress that decreases plant growth,ranking among the leading factors that limit agriculturalproductivity. High concentrations of NaCl can arrest plantdevelopment and result in plant death. Salt stress triggers severalinteracting events in plants, including the inhibition of enzymaticactivity in metabolic pathways, decreased uptake of nutrients inroots, decreased efficiency of carbon use, and the denaturation ofprotein and membrane structures.The Salt Overly Sensitive (SOS) pathway is thought to play

essential roles in conferring salt tolerance in Arabidopsis thaliana.SOS3, the first gene derived from this family that was cloned, wasobtained throughgenetic screening of salt-sensitivemutants andmap-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, aplasma membrane Na+/H+ antiporter (Shi et al., 2000; Qiu et al.,2002).AlthoughCa2+ is not required forSOS2 andSOS3 to interact inthis process, Ca2+ is thought to increase the phosphorylation of thesynthesized peptide substrate p3 through the SOS2–SOS3 complex(Halfter et al., 2000). SCABP8/CBL10 as a putative Ca2+ sensorinteracts with the protein kinase SOS2 to protect Arabidopsis shootsfrom salt stress by modulating ion homeostasis (Quan et al., 2007;Kim et al., 2007). Similar functions in biochemical and cellular testsare fulfilled by SCABP8 and SOS3; however, they must also performdistinct regulatory functions in the salt stress response because theycannot replace each other in genetic complementation experiments,for example (Quan et al., 2007).

The mutations in SOS1, SOS2 and SOS3 all decrease the Na+/H+exchange activity, and a constitutively active SOS2 enhancesNa+/H+exchange activity in an SOS1-dependent and SOS3-independentmanner (Qiu et al., 2002). The root and shoot growth of sos1 and sos2mutants is more significantly inhibited than that of sos3 under mildsalt stress (Zhu et al., 1998). However, SCABP8/CBL10 mainlyprotects Arabidopsis shoots from salt stress (Quan et al., 2007; Kimet al., 2007). Consistent with their function, the expression of SOS1and SOS2 is detected in both root and shoot tissues, whereas theexpression 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 ofthe APETALA2 (AP2)-domain family, has been found only in plantsystems (Mizukami and Fischer, 2000). Like AP2, ANT containstwo AP2 domains that are homologous with the DNA-bindingdomain of ethylene-response-element binding proteins (Mizukamiand Fischer, 2000). ANT as a transcription factor is stronglyexpressed in many dividing tissues in the plant, in which it playscentral roles in developmental processes, such as shoot and flowermeristem maintenance, organ size and polarity, flower initiation,ovule development, floral organ identity, as well as cell proliferation(Horstman et al., 2014).

Here, we report that ANT mainly protects Arabidopsis shootsfrom salt stress and show that SCABP8 is a downstream target ofANT in the regulation of salt stress because the mutation of SCABP8suppresses the ant-knockout salt-tolerant phenotype, implying thatSCABP8 plays a predominant role in inhibiting ANT-induced salttolerance. Thus, ANT is a novel regulator of salt stress, binding tothe SCABP8 promoter in order to mediate salt tolerance.

RESULTSANT expression patternsThe Arabidopsis ANT gene, encoding an AP2-like transcriptionfactor, controls plant organ cell number and organ size throughoutshoot development (Mizukami and Fischer, 2000). Usinghistochemical GUS staining to detect expression of GUS underthe ANT promoter (ANT-pro:GUS), ANTwas mainly detected in theReceived 25 March 2015; Accepted 1 June 2015

1School of Bioengineering and Biotechnology, Tianshui Normal University, Tianshui 741001, People’s Republic of China. 2Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, People’s Republic of China. 3Division of Molecular Life Sciences, Pohang University of Science and Technology, Hyoja-dong, Pohang, Kyungbuk 790-784, Republic of Korea.

*Authors for correspondence (menglaisheng@mail.kib.ac.cn; ybwang2015@yeah.net; liuaizhong@mail.kib.ac.cn)

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tissues undergoing active division (including young leaf blades, leafveins, stem veins, stamens, pistils, meristem tissue and vascularcylinder) but not in mature tissue or root tissue (including the maturezone of roots and mature leaves) (Fig. 1A–H; also see Mizukamiand Fischer, 2000; Elliott et al., 1996; Klucher et al., 1996). In theroots, ANTwas only expressed in the root tips and vascular cylinder(Fig. 1G,H). Thus, it appears that ANT mainly functions in shoottissue; this result is consistent with the role of ANT in controllingplant organ cell number and organ size throughout shootdevelopment (Mizukami and Fischer, 2000). In this work, wefound that the ant-knockout mutant, but not plants ectopicallyexpressing ANT under the control of the constitutive 35S promoter(35S:ANT), exhibited a tolerant phenotype under high salt stress(Fig. 2). To examine whether the expression of ANT is induced bytreatment with NaCl, total RNAwas extracted from wild-type plantsthat had been treated with 75 mM NaCl or 100 mM NaCl for 3 and6 h. The transcription of ANT was substantially lower at 3 and 6 hafter treatment in comparison with that of the control plants(supplementary material Fig. S1A).

ant-knockout and 35S:ANT show different responses to highsodium salt stressTo further determine whether ANT is involved in the regulation ofthe salt stress response, ant-knockout (stock, SALK-022770) and35S:ANT plants were analyzed. Reverse-transcriptase (RT)-PCR

analyses indicated that the 35S:ANT lines over-accumulated ANTtranscripts compared with the wild type, whereas ANT had beeneliminated in the ant-knockout (SALK-022770) lines (seesupplementary material Fig. S1B).

Seeds of the ant-knockout mutant, 35S:ANT and wild-type (Col-0)lines were sown on solid Murashige and Skoog (MS) medium with100 mM NaCl for 30 days. The salt tolerance of the ant-knockoutplants wasmaintained throughout their development from seedlings tomature plants, whereas a number of 35S:ANT transgenic plants weredry and dead compared with the wild type (Col-0) (supplementarymaterial Fig. S2A). The ant-knockout exhibited longer primary roots,whereas the 35S:ANT plant showed shorter primary roots than thewildtype (supplementary material Fig. S2A,B). The number of ant-knockout lateral roots was increased, whereas the number of 35S:ANTlateral roots was not different to that of the wild type (supplementarymaterial Fig. S2A,C). Consequently, although the overall weight offresh roots of the ant-knockout mutant was only ∼1.6 times greaterthan that of the wild type (supplementary material Fig. S2E), theweight of fresh shoots in the ant-knockout mutant was increased∼2.9 times over that of thewild type (Col-0), and its leaves were alsolarger (supplementary material Fig. S2A,D). However, the seedlingweight of ant-knockout, 35S:ANT and wild-type plants was notdifferent on medium without salt (supplementary material Fig. S3).

To further characterize the salt insensitivity of the 35S:ANT andant-knockout mutant plants, 5-day-old seedlings were transferred tosolid MS medium with NaCl (75 mM NaCl and 100 mM NaCl) for15 days. On solid MS medium with both 75 mM NaCl and 100 mMNaCl, the length of the ant-knockout primary roots was increased,whereas the length of the 35S:ANT primary roots was not different tothat of the wild type (Fig. 2A,B). OnMSmediumwith 75 mMNaCl,the number of ant-knockout lateral roots increased, whereas thenumber of 35S:ANT lateral roots was not significantly different to thatof thewild type (Fig. 2A,C). However, onMSmediumwith 100 mMNaCl, the number of ant-knockout lateral roots increased, whereas thenumber of 35S:ANT lateral roots was reduced relative to that of thewild type (Fig. 2A,C). Consequently, the overall weight of fresh rootsin the ant-knockout mutant increased relative to that of the wild type(Col-0) by only ∼1.16- and ∼1.22-fold on MS medium with 75 mMNaCl and 100 mM NaCl, respectively (Fig. 2A,E), whereas theweight of fresh shoots in the ant-knockout mutant was ∼1.44 and∼1.53 times greater on MS medium with 75 mM NaCl and 100 mMNaCl, respectively (Fig. 2A,D). Moreover, the weight of 35S:ANTfresh shoots was decreased on MS medium with 100 mM NaCl(Fig. 2A,D).However, the seedlingweight ofant-knockout,35S:ANTand wild-type plants was not different on medium that lacked salt(supplementary material Fig. S3).

To confirm that ANT is involved in the regulation of salt stress, weassayed whether transforming the mutant with ANT genomicDNA (ant-knockout+35S:ANT) could render the ant-knockout lesssensitive to salt, similar towild type. Five-day-old ant-knockout, ant-knockout+ 35S:ANT, 35S:ANT and Col-0 seedlings were transferredto solid MS medium with 100 mM NaCl for 10 days. On solid MSmedium without NaCl, the weight of the 15-day-old shoots did notdiffer (supplementary material Fig. S3A,B). However, on MSmedium with 100 mMNaCl, the weight of 15-day-old ant-knockoutshoots increased, whereas that of corresponding 35S:ANT plantsdeclined (supplementary material Fig. S3A,B). Furthermore, theweight of shoots of ant-knockout+35S:ANT and Col-0 seedlings didnot differ (supplementary material Fig. S3A,B), indicating that theant-knockout mutant phenotype can be rescued.

Taken together, our findings indicate that ANT functionspredominantly in the shoot response to salt toxicity.

Fig. 1. ANT expression analysis. Expression of ANT-pro:GUS in 4-day-oldleaf blades (A), veins (B), pollen and stamens (C), stem veins (D), siliques (E),pistils (F), root mature zone (G) and root tips (H). Scale bars: 2 mm (A,E);100 μm (B–D,F–H).

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The growth defects of ant-knockout and 35S:ANT seedlingsare not specific to the Na+ responseTo determine whether the growth defects of the ant-knockoutmutant and 35S:ANT seedlings are specific to the Na+ response;these seedlings were treated with a different kinds of salt. Upontreatment with 100 mM KCl, ant-knockout and 35S:ANT seedlingswere less sensitive and more sensitive relative to wild type,respectively (Fig. 3A–D). Similarly, upon treatment with 10 mMLiCl, ant-knockout mutant and 35S:ANT seedlings also appeared tobe less sensitive and more sensitive relative to wild type,respectively (Fig. 3A–D). Furthermore, ant-knockout mutant and35S:ANT seedlings that had been treated with a combination of both100 mM KCl and 10 mM LiCl exhibited less-sensitive and more-sensitive phenotypes relative to those observed upon singletreatment with 100 mM KCl or 10 mM LiCl, respectively(supplementary material Fig. S4). However, the weight of ant-knockout, 35S:ANT and wild-type seedlings did not differ onmedium that lacked salt (supplementary material Fig. S4).

ANT negatively modulates SCABP8 at the level oftranscriptionTo detect the downstream target genes of ANT, the expressionlevels of the genes involved in the salt stress response wereanalyzed by using quantitative RT-PCR analyses. The SOSpathway plays an essential role in the salt tolerance of

Arabidopsis thaliana. Thus, SOS1, SOS2, SOS3 and SCABP8were the first genes examined. Transcripts of SCABP8 were muchmore abundant in ant-knockout plants, whereas transcripts ofSCABP8 were lower in 35S:ANT plants compared with those in thewild type (Fig. 4A). We also observed that the expression levels ofSOS1, SOS2 and SOS3 were not significantly different betweenant-knockout, 35S:ANT and wild-type plants (Fig. 4A). Moreover,ANT transcript abundance was similar between wild-type, sos1,sos2, sos3 and scabp8 plants (Fig. 4B). Therefore, ANT negativelymodulates SCABP8 expression at the transcript level. Plant NHX1,NHX4, NHX5 and NHX6 transporters are involved in salttolerance (Hernandez et al., 2009; Bassil et al., 2011). Thesefindings demonstrate that the expression levels of the genesencoding these transporters were not altered in 35S:ANT seedlingsrelative to those in wild type (Fig. 4G), indicating that ANTspecifically regulates SCABP8.

ANT associates with the SCABP8 promoter both in vitro andin vivoTo investigate the binding of ANT to the SCABP8 promoter, weperformed nucleotide sequence analysis and found that the two AP2domains in ANT were selectively associated with the consensussequence 5′-gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t)-3′ (lowercaseletters indicate somewhat less-conserved positions that are presentin at least 65% of the selected sites) (Nole-Wilson and Krizek, 2000;

Fig. 2. ant-knockout seedlings show resistance to salt stress.Seedlings were grown on solid MSmediumwithout NaCl for 5 days andwere then transferred tosolid MSmedium with 75 mMNaCl or 100 mMNaCl for 15 days. (A) Representative 20-day-old ant-knockout (ant-KO), wild-type (Col-0) and 35S:ANT seedlingsgrown on solid MS medium with 75 mM NaCl or 100 mM NaCl for 15 days. The magnification is the same for each image. (B–E) Bar graphs showing thedifferences in primary root length (B), lateral root number (C), the weight of fresh shoots (D) and of fresh roots (E) between the 20-day-old ant-knockout, 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 s.d.; **P<0.01, *P<0.05; n=15.

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Krizek, 2003). Our nucleotide sequence analysis also found that thepromoters of the SCABP8 gene contained conserved sequencemotifs (Fig. 5 and Fig. 4C). This portion of ANT corresponds toamino acid residues 276–456 of full-length ANT, comprising thetwo AP2 repeats and the linker region between them, and will bereferred to as ANT-AP2R1R2 (Nole-Wilson and Krizek, 2000).Glutathione S-transferase (GST) was fused to the DNA-bindingdomain of ANT-AP2R1R2, and the protein was expressed inEscherichia coli and purified by using the GST tag. A gel mobilityshift assay was performed to assay whether the recombinant ANT-AP2R1R2 protein could associate with the SCABP8 promoter.When the ANT-AP2R1R2 protein and the probes to the ANT-binding consensus sequences were added, a shift was detected as aresult of DNA binding; no band was detected when only the probesto the ANT-binding consensus sequences were added (Fig. 5A,B).When unlabeled probes of the ANT-binding consensus sequenceswere added to the reaction mixture, the DNA-binding was abolished(Fig. 5B). Furthermore, the GST–ANT-AP2R1R2 protein wasunable to bind to mutated DNA probes (Fig. 5C,D). These dataindicate that the ANT-AP2R1R2 DNA-binding site binds to theSCABP8 promoter in vitro.To further investigate the possible binding in vivo, we used

transgenic shoots expressing ANT with a C-terminal hemagglutinin(HA) tag under the control of the constitutive 35S promoter (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 ofthe S2 (−1152 to −852 bp) or S3 (−348 to −48 bp) regions(Fig. 4D,E). These data clearly indicate that ANT binds to theSCABP8 promoter in vivo, which is required to suppress SCABP8expression.

ANT acts genetically upstream of SCABP8To assay whether ANT acts genetically upstream of SCABP8 inregulating the salt stress response, we performed a double-mutantanalysis by combining ant-knockout with scabp8 lines (to generateant-knockout-scabp8) that were insensitive and supersensitive tohigh salt stress, respectively. We selected the genotypes fromthe segregating F3 population by PCR genotyping. Mutationof SCABP8 significantly suppressed the insensitive phenotype ofthe ant-knockout line under high salt stress (Fig. 6). The lengthof the ant-knockout primary roots obviously increased, whereas thelengths of the ant-knockout-scabp8 and scabp8 primary roots weresignificantly reduced relative to that of the wild type on solid MSmedium with NaCl (75 mM NaCl and 100 mM NaCl) (Fig. 6A,B).The overall weights of fresh shoots and roots in the ant-knockoutmutants were increased, whereas those in the ant-knockout-scabp8and scabp8mutants were decreased relative to those of the wild typeon MS medium with 75 mM NaCl and 100 mM NaCl, respectively(Fig. 6A,C,D). Taken together, the above results indicate that ANTacts genetically upstream of SCABP8 in regulating the salt stressresponse (Fig. 7).

Fig. 3. ant-knockout and 35S:ANT seedlings showdifferent responses from thewild type (Col-0) to KCl or LiCl.Seedlings were grown on solidMSmediumwithout 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-knockout(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-knockout(d), wild-type (Col-0; e) and 35S:ANT (f ) seedlings grown on solid MS medium with 10 mM LiCl for 15 days. The magnification is the same for each image.(B–D) Bar graphs showing the differences in primary root length (B), lateral root number (C) and the weight of fresh shoots (D) between the 20-day-oldant-knockout, wild-type (Col-0) and 35S:ANT seedlings grown on solid MS medium with 100 mM KCl or 10 mM LiCl. Error bars represent s.d.; *P<0.05;n=15. Scale bars: 2.0 cm (Aa–Af).

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DISCUSSIONANT, encoding a transcription factor of the APETALA2 family, isinvolved in cell proliferation and growth control (Elliott et al., 1996;Klucher et al., 1996; Eshed et al., 1999; Krizek, 1999; Mizukamiand Fischer, 2000; Nole-Wilson and Krizek, 2000). Here, we haveshown that ant-knockout mutants exhibit a salt-tolerant phenotype,whereas transgenic plants harboring the 35S:ANT construct exhibita more sensitive phenotype under high salt stress. Under normalgrowth conditions in the glasshouse, 35S:ANT exhibited a greatercell number and larger organ size (seeds, leaves, flowers, large rootsystems and higher weight) (Mizukami and Fischer, 2000), at thesame time, 35S:ANT plants were hypersensitive to salt stress. Thus,

a regulator of plant development might be involved in modulatingenvironmental stress. Arabidopsis AN3 (also known as GIF1), atranscription 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 have found that AN3 controlswater-use efficiency (WUE) and drought tolerance by regulatingstomatal density and improving root architecture through the trans-repression of YODA (YDA) (Meng and Yao, 2015). The ArabidopsisE3 SUMO ligase SIZ1 regulates both plant growth and droughttolerance, that is, the null mutant siz1-3 shows smaller organ sizeowing to reduced expression of genes involved in brassinosteroidbiosynthesis and signaling, at the same time, mutant seedlings of

Fig. 4. ANT negatively regulates SCABP8 at the transcript level. (A) Bar graph showing the differences in the expression levels of SOS1, SOS2, SOS3and SCABP8 in 15-day-old ant-knockout (ant-KO), wild-type (Col-0) and 35S:ANT seedlings grown under white-light conditions. ***P<0.001, *P<0.05; n=3. Atleast five independently propagated lines were used for each seedling type, and the expression of the genes in wild type was set as 1.0. Quantification wasnormalized to the expression ofUBQ5 (also known asRPS27AC); n=3. (B) Bar graph showing the differences in the expression ofANT between wild-type (Col-0),sos1, sos2, sos3 and scabp8 seedlings grown under white-light conditions. (C) Schematic of theSCABP8 promoter loci (at4g33000) and their amplicons for ChIPanalysis. Several amplicons in the SCABP8 promoter were used for ChIP analyses. Additionally, S1 contains the ANT-associating consensus sequences[5′-gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t)-3′]. S1, S2 and S3 indicate three different regions in the SCABP8 promoter. The nucleotides in blue denote theconserved loci targeted by ANT. TGA-1690 indicates that there are 1690 bp between the TGA stop codon of at4g32990 and the ATG start codon of at4g33000 ofSCABP8. (D,E) ChIP analysis. Enrichment of particular SCABP8 chromatin regions with antibodies against HA (as a control) or GFP in 35S:ANT:HA transgenicshoots (E) and roots (F), as detected by real-time PCR analysis. Quantification was normalized to the expression of UBQ5; n=3. Input was set as 100%.(G) Expression of NHX1, NHX4, NHX5 and NHX6 in Col-0 and 35S:ANT seedlings. Bar graph showing the differences in the expression levels of NHX1, NHX4,NHX5 and NHX6 in 15-day-old wild-type (Col-0) and 35S:ANT seedlings grown under white-light conditions; n=3. At least five independently propagated lineswere used for each seedling type, and the expression of the genes in the wild type was set as 1.0. Quantification was normalized to the expression ofUBQ5; n=3.All error bars represent s.d.

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siz1-3 exhibit significantly lower tolerance to drought stress(Catala et al., 2007). Loss-of-function of PROTEIN S-ACYLTRANSFERASE10 (PAT10) leads to pleiotropic growth defects,including smaller leaves, dwarfism and sterility, whereas pat10mutants exhibit hypersensitivity to salt stresses (Zhou et al., 2013).NHX1 (K+/H+) and NHX2 are vacuolar proteins, and they controlvacuolar pH and K+ homeostasis, and salt tolerance (Bassil et al.,2011). Thus, the role of NHX1 and NHX2 is to regulate K+/H+

exchange, and this exchange modulates cell expansion in rapidlyelongating tissues – for example, filaments and hypocotyls – andplays unique and specific roles in flower development (Bassil et al.,2011). We speculate that, similar to NHX1 and NHX2, ANT isinvolved in regulating either plant growth or development throughregulation of the SOS system, in which SCABP8/CBL10 as aputative Ca2+ sensor interacts with the protein kinase SOS2 toprotect Arabidopsis shoots from salt stress by modulating ionhomeostasis (Quan et al., 2007; Kim et al., 2007). The connectionbetween plant development and environmental stress could open upfuture research to provide further insights into the mechanism thatlinks development and stress.On MS medium supplemented with 100 mM NaCl, sos3, 35S:

ANTand scabp8mutants exhibited severe growth suppression in bothshoot and root tissues; however, nearly complete growth arrestoccurred only in the roots of sos3 and the shoots of 35S:ANT andscabp8 plants (Zhu et al., 1998; Quan et al., 2007; Fig. 2). At a lowerNaCl concentration (75 mM), the root growth in the sos3mutantswassignificantly suppressed (Zhu et al., 1998), whereas the shoot growthin the 35S:ANT and scabp8mutants was more inhibited (Quan et al.,2007; Fig. 2). These findings tell us that salt tolerance in plants

under extreme conditions is necessary for the functional integrationof all tissues but also that SOS3, ANT and SCABP8 are needed forsalt tolerance in a tissue-specific manner. Their respectiveexpression patterns were consistent with these findings, asassayed by using promoter–GUS fusions (Fig. 1; also see Quanet al., 2007). SOS3was only expressed in root tissues, particularly inroot tips, whereas ANT and SCABP8 were preferentially expressedin shoot tissues. Thus, SCABP8 and ANT mainly protect shoottissues, whereas SOS3 protects root tissues from salt stress,suggesting that tissue specificity could be important for plantscoping with environmental stress.

Although the expression of SCABP8 was upregulated upon saltstress, the expression ofANTwas downregulated, indicating thatANTand SCABP8 differ in their transcriptional regulation in response tosalinity. Functional analyses, as well as the specific Na+ sensitivity ofthe sos1, sos2, sos3 and scabp8 mutants, indicate that SOS3 andSCABP8have overlapping functions in the activation of SOS2and itsdownstream target, SOS1 (Fig. 7; also see Quan et al., 2007). Thus,we conclude that ANT directly binds to the SCABP8 promoter inorder to regulate the SOS signaling cascade in the salt stress response.

MATERIALS AND METHODSPlant materials and growth conditionssos1, sos2, sos3 and scabp8 mutants in the Col-0 background have beendescribed previously (Quan et al., 2007). ant-knockout (SALK-022770),ant-knockout+35S:ANT and 35S:ANT seeds (Ph2GW7; Col-0 background;homozygote; hygromycin) were kindly provided by Professor H. G. Nam[Daegu Gyeongbuk Institute of Science and Technology (DGSIT), Korea].ant-knockout (SALK-022770) is a T-DNA mutant, the single-pass

Fig. 5. Gel-shift analysis. (A,C) Gel-shift analysis was used to investigate association of the ANT protein with cis-elements in the promoter of SCABP8. Theoligonucleotide sequences of the probe and themutated form of the probe (mProbe) within theSCABP8 promoter used in the EMSA are shown. Underlined lettersin blue indicate the sequences of the ANT-recognition motifs. mProbe – ANT-recognition motifs in the probe were mutated, as indicated by capital letters.(B) Unlabeled SCABP8 promoter (2 µg) was used as a competitor to determine the specificity of the DNA-binding activity of ANT. (D) The mutant version ofthe SCABP8 promoter (CC changed to GG) was labeled with biotin and used for EMSA with ANT polypeptides. Arrow indicates ANT-AP2R1R2–probecomplexes, * indicates free probe.

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sequence of which was recovered from the left border of the T-DNA; andthis sequence lies within 300 bases of the 3′-end of At4g37750, as has beendescribed by Arabidopsis Biological Resource Center (ABRC). Transgenicplants were generated using the Agrobacterium-tumefaciens-mediated floraldip method (Zhang et al., 2006). For ant-knockout and 35S:ANT

identification, the primers forward 5′-ATGAAGTCTTTTTGTGATAAT-GA-3′ and reverse 5′-TTCAATCTCTTTCTGATAATTCTC-3′ were used.ant-8 (CS3944) was obtained from ABRC.

Double ant-knockout-scabp8 mutants were generated through geneticcrosses, and homozygous lines were identified through comparison withthe parental phenotype and PCR-based genotyping. The ANT-gene-specificprimers were forward 5′-GAGAAGGAAGAGCAGTGGTTTCT-3′ andreverse 5′-CAGATGTTCGGATCCAATATGG-3′; the SCABP8 gene-specific primers have been described previously (Quan et al., 2007).

Plants exhibiting the 35S:ANT phenotype [large rosette leaves and largeseeds grown under white light (Mizukami and Fischer, 2000)] in the F2populations were screened for Dr5:GUS expression in roots. F3 seeds werecollected from those exhibiting expression, and lines expressing GUS in allF3 plants were used for subsequent analysis.

The seeds were subjected to 4°C for 3 days and then sown onto solid MSmedium that had been supplemented with 1% sucrose. The roots, seeds,leaves and embryos were photographed, and then they were analyzed usingImageJ software. The seedlings grown on agar were maintained in a growthroom under 16-h–8-h light–dark cycles with cool white fluorescent light at21°C±2°C. Plants grown in soil-less medium were maintained in acontrolled-environment growth room under 16-h–8-h light–dark cycleswith cool white fluorescent light at 21°C±2°C.

Salt sensitivitySeeds of the mutant and wild-type (Col-0) plants were sterilized in asolution containing 30% sodium hypochlorite and 0.1% Triton X-100 for10 min, washed five times with sterilized water, and sown on MS mediumwith 0.8% Phytagel (Sigma-Aldrich). The plates were placed at 4°C for3 days, and the seeds were then germinated vertically at 21°C±2°C undercontinual illumination. Five-day-old seedlings with a root length of

Fig. 6. scabp8mutation suppresses the ant-knockout mutant phenotype. (A) Seedlings were grown on solid MS medium without NaCl for 5 days and werethen 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 theindicated 20-day-old seedlings grown on solid MSmediumwith 75 mMNaCl or 100 mMNaCl; n=12. (C) Bar graph exhibiting the differences in theweight of freshshoots between the indicated 20-day-old seedlings grown on solid MSmedium with 75 mMNaCl or 100 mMNaCl; n=12. (D). Bar graph exhibiting the differencesin root freshweight between the indicated 20-day-old seedlings grown on solid MSmediumwith 75 mMNaCl or 100 mMNaCl. Error bars represent s.d.; **P<0.01,*P<0.05; n=12. ant-KO, ant knockout; ant-KOscabp8, ant-KO-scabp8; WT, wild type.

Fig. 7. Salt stress induces downregulation of ANT. ANT associates directlywith the SCABP8 promoter and causes the upregulation of SCABP8, therebyinducing salt stress tolerance in the plant. In vitro, SOS3 physically interactswith and activates a Ser/Thr protein kinase, SOS2. Among the downstreamtargets of the SOS3–SOS2 complex is SOS1, a plasma-membrane–localizedNa+/H+ antiporter (Shi et al., 2000; Qiu et al., 2002; Quintero et al., 2002).SCABP8/CBL10, a putative Ca2+ sensor, interacts with the protein kinaseSOS2 to protect Arabidopsis shoots from salt stress (Quan et al., 2007).

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∼1.5 cm were transferred onto MS medium with salt added as previouslydescribed (Quan et al., 2007).

RT-PCRTotal RNA was extracted from the tissues indicated in the figuresusing TRIZOL reagent (Invitrogen), as has been previously described byYu et al. (2008). SYBR green was used to monitor the kinetics of the PCRproducts in the real-time RT-PCR analyses, as has been previouslydescribed by Yu et al. (2008). To analyze the expression levels of SOS1,SOS2, SOS3 and SCABP8 in Col-0, ant-knockout and 35S:ANTseedlings, the following primers were used – forward 5′-ATGACGACT-GTAATCGACGCGAC-3′ and reverse 5′-CGAATTCCATGGCCGATCT-TTC-3′ for SOS1; forward 5′-ATGACAAAGAAAATGAGAAGAGT-3′and reverse 5′-CACAAACTCCAAAACTATATAT-3′ for SOS2; forward5′-TGCAATGCGACCACCGGGAT-3′ and reverse 5′-TTTCATGGACC-GGCGCGCTT-3′ for SOS3; forward 5′-ATTGCCGCAGCACATCGCCT-3′ and reverse 5′-TGGAGCTTGGAACAGCGCCAG-3′ for SCABP8. Toanalyze the expression levels of NHX1, NHX4, NHX5 and NHX6 in Col-0and 35S:ANT seedlings, the following primers were used – forward 5′-ATGTTGGATTCTCTAGTGTCGA-3′ and reverse 5′-ACGAAATTGCG-GAAAAACTGCTT-3′ for NHX1; forward 5′-ATGGTGATCGGATTAA-GCACAAT-3′ and reverse 5′-TGGAAGAAGATAAATAAAGAAGAG-3′for NHX2; forward 5′-ATGGAGGAAGTGATGATTTCTC-3′ and reverse5′-TTTAGGTTGAAGACTGAAACCTG-3′ for NHX5; forward 5′-ATG-TCGTCGGAGCTGCAGAT-3′ and reverse 5′-GAAAGAAGAACTCATCG-TGGAAA-3′ for NHX6.

To analyze ANT expression in Col-0, sos1, sos2, sos3 and scabp8seedlings, the primers forward 5′-AGGTGGCAAGCACGGATTGGT-3′and reverse 5′-AGGCAACGCGAAAATCGCCG-3′ were used. To analyzethe expression levels of ANT in Col-0, ant-knockout and 35S:ANTseedlings, the following primers were used – forward 5′-TCTTCTTCTG-TTCCACCTCAAC-3′, reverse 5′-TCTGCAGCTTCTTCTTGGGTT-3′;and forward 5′-CTTAGGAGAAGGAAACACTG-3′, reverse 5′-AATAC-GTCGGCGATTCTCCG-3′ were used for TUB4.

Plasmid constructsFor the ANT promoter analysis, the promoter–GUS construct At4g37750 wascreated by inserting approximately2.0-Kbpromoter fragments intopCB308R,as previously described byLei et al. (2007). The primers used forANTwere P15′-ggggacaagtttgtacaaaaaagcaggctGTTCAATAAATATGTTTCCACGTA-3′and P2 5′-ggggaccactttgtacaagaaagctgggtCGCTTTTAGCCCTACAAATT-CC-3′. To obtain theANT:HAplasmid (CB2004),we used the primers forward5 ′-ggggacaagtttgtacaaaaaagcaggctTTCAATCTCTTTCTGATAATTCTC-3′and reverse 5′-ggggaccactttgtacaagaaagctgggtTCAAGAATCAGCCCAAG-CAGC-3′. Lowercase letters denote sequences from the connector primers,whereas capital letters denote the sequences of targeted genes.

GUS stainingUsing a buffer mix [1 mM X-gluc, 60 mM NaPO4 buffer, 0.4 mM ofK3Fe(CN)6/K4Fe(CN)6 and 0.1% (v/v) TritonX-100], the samples (transgenicplants harboring and expressing ANT-Pro:GUS) were stained and thenincubated at 37°C for 8 h.After GUS staining, chlorophyll was removed usingwashes of 30%, 50%, 70%, 90% and 100% ethanol for approximately 30 mineach.GUSstainingwasperformedaspreviouslydescribedbyLei et al. (2007).

ChIP assayTransgenic lines overexpressing 35S:ANT:HAwere used in this assay. ChIPwas performed as previously described (Yoo et al., 2010). HA- and greenfluorescent protein (GFP)-tag-specific monoclonal antibodies were used forChIP analysis. The ChIP DNA products were analyzed by using quantitativeRT-PCR with primers that had been designed to amplify ∼300-bp DNAfragments in the promoter region of SCABP8. The following primersequences were used – S1 P1 5′-ATAAACAATTTTAATCTCGATA-3′, P25′-ATTTTGCGGTATATTTTGTTATT-3′; S2 P3 5′-AAAATAGACAAC-AAATTCTTTT-3′, P4 5′-AATCATAATATATGACCTTTGA-3′; S3 P5 5′-GTTGAGATTCCAACTAGTATAT-3′, P6 5′-TGTCTTATCAAACTCTA-TAGTTG-3′.

Protein expression and purificationThe plasmid pGEX-5X-1 encoding ANT was used in this experiment. Thecoding sequence of ANT was amplified using the primer pair (5′-GGATCC-ATGAAGTCTTTTTGTGA-3′ and 5′-GAATTCAGAATCAGCCCAAGC-3′) and cloned into the BamHI and EcoRI restriction sites of the pET28aplasmid to generate the final plasmid. Recombinant GST-tagged ANT wasextracted from transformed E. coli (Rosetta2) after 10 h of incubation at 16°Cfollowing induction with 10 µM isopropyl β-D-1-thiogalactopyranoside.These recombinant proteins were purified using GST–agarose affinity.

Electrophoretic mobility shift assayThe electrophoresis mobility shift assay (EMSA) was performed usingthe LightShift Chemiluminescent EMSA Kit (Pierce, 20148) accordingto the manufacturer’s instructions. The biotin-labeled DNA fragments(5′-GAACCGACCAGTTAACCGATAATTATACATCCGGTTTGACTT-ACCCATAATTTATCCAAAACTAATCCGATTTTGA-3′) and mutatedDNA fragments (5′-GAAGGGACCAGTTAAGGGATAATTATACATC-CGGTTTGACTTACCCATAATTTATCCAAAACTAATGGGATTTTG-A-3′) were synthesized, annealed and used as probes, and unlabeled versionsof the sameDNA fragmentswere used as competitors in this assay. The probeswere incubated with the ANT-fused protein at room temperature for 15 min inbindingbuffer (50 mMHEPES-KOH,pH7.5; 375 mMKCl; 6.25 mMMgCl;1 mM DTT; 0.5 mg/ml BSA; glycerol 25%). Each 20 ml binding reactioncontained 25 fmol biotin-labeled probes and 5 mg protein, and 1 mg Poly(dIdC) was supplemented into the reaction to minimize nonspecificinteractions. And then, we analyzed the reaction products by using 8.0%native polyacrylamide gel electrophoresis. Electrophoresis was performed at100 V for approximately 1–2 h in TGE buffer (containing 12.5 mM Tris;95 mM glycin; and 0.5 mM EDTA, pH 8.3; precooled at −12°C). The DNAfragments on the gel were transferred onto a nitrocellulose membrane with0.5× TBE at 100 V (∼400 mA) for 50 min at 4°C. The DNAwas cross-linkedonto the membrane, and the membrane was incubated in the blocking bufferfor 20–30 min with gentle shaking; the membrane was then transferred into ablocking buffer, which comprised 33.3 ml of stabilized Streptavidin–horseradish-peroxidase conjugate with 10 ml of blocking buffer, accordingto the manufacturer’s protocol. The membrane was washed 4–6 times for5 min eachwith awashing buffer. Biotin-labeledDNAwas detected using thechemiluminescence method according to the manufacturer’s protocol.

AcknowledgementsWe thank Professor Hong Gil Nam (DGIST, Korea) and Cheng-Bin Xiang(University of Science and Technology of China, China) for their support during theinitial stages of this work. ant-knockout (SALK-022770) and 35S:ANT were kindlyprovided by Professor H. G. Nam (DGSIT, Korea). sos1, sos2, sos3 and scabp8seeds were kindly provided by Professor Y. Guo (Agricultural University of China,China).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsL.-S.M. designed experiments. L.-S.M. performed the experiments. L.-S.M., S.-Q.Y.and Y.-B.W. completed statistical analysis of data. L.-S.M. and A.L. wrote, edited andrevised this manuscript.

FundingWe are grateful for National Key Basic Research Program of China [grant number2014CB954100]; and National Key Technology R&D Program [grant number2015BAD15B02]. This research was also supported by ‘QingLan’ TalentEngineering Funds provided by Tianshui Normal University.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.172072/-/DC1

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