Gene 552 (2014) 146–154

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Methods paper

Overexpression of CaDSR6 increases tolerance to drought and saltstresses in transgenic Arabidopsis plants

Eun Yu Kim a, Young Sam Seo b, Ki Youl Park a, Soo Jin Kim a,1, Woo Taek Kim a,⁎a Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Koreab National Resources Research Institute, Korea Ginseng Corp., Daejeon 305-805, Republic of Korea

Abbreviations: CaMV, cauliflowermosaic virus; CRWLdifferential display polymerase chain reaction; DSR6, drfreshweight; qRT-PCR, quantitative reverse transcription p⁎ Corresponding author.

E-mail address: wtkim@yonsei.ac.kr (W.T. Kim).1 Current address: Department of Horticultural Crop

Horticultural and Herbal Science, Rural Development AdRepublic of Korea.

http://dx.doi.org/10.1016/j.gene.2014.09.0280378-1119/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 June 2014Received in revised form 11 September 2014Accepted 14 September 2014Available online 16 September 2014

Keywords:Arabidopsis thalianaAsp-rich domainCaDSR6-overexpressing transgenic plantsHot pepperDrought and salt stress tolerance

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as adrought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to droughtstress tolerancewas unknown. In this report, full-lengthCaDSR6 cDNAwas isolated. The deduced CaDSR6proteinwas composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in itscentral region. This Asp-rich domainwas highly conserved in all plant DSR6 homologs identified and shared a se-quence identitywith theN-terminal regions of yeast p23fyp andhumanhTCTP,which contain Rab protein bindingsites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, asidentified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFPleaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehy-dration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B,RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild typeplants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is asso-ciated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype intransgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response todrought and salt stresses.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Environmental abiotic stresses, such as drought, high salinity, heavymetals, and extreme temperatures, hinder the growth anddevelopmentof soil plants resulting in serious crop losses at a global level. Higherplants develop a number of genetic and cellular strategies to copewith detrimental stress conditions (Zhu, 2002; Shinozaki et al., 2003;Tardieu et al., 2011; Verslues and Juenger, 2011). Diverse sets of genesexpressed in response to abiotic stress have been identified, and eluci-dation of their physiological or cellular roles in terms of either stress-tolerance or sensitivity is a critical issue in current plant biology(Ahuja et al., 2010; Huang et al., 2012; Jirschitzka et al., 2013). Thus, itis essential to study the functions of stress-responsive genes to

, cut rosettewater loss; DD-PCR,ought stress responsive 6; FW,olymerase chain reaction.

Research, National Institute ofministration, Suwon 441-440,

ameliorate stress tolerance in crop plants (Mittler and Blumwald,2010; Deikman et al., 2012; Cabello et al., 2014; Ismail et al., 2014).

Hot pepper (Capsicumannuum L.) is a solanaceous species closely re-lated to tomato and tobacco plants. Hot pepper is one of the most eco-nomically valuable crops that has been cultured worldwide for its hot-taste fruits (Aguilar-Meléndez et al., 2009; Bosland and Votava, 2012).We previously conducted subtractive hybridization and differential dis-play polymerase chain reaction (DD-PCR) experiments and isolated abroad spectrum of partial cDNA clones that were rapidly induced bydrought stress in the root and leaf tissues of hot pepper plants (Parket al., 2003; Hong and Kim 2005). Among those are CaLEAL1 (Parket al., 2003), CaDREB1 (Hong and Kim 2005), CaPUB1 (Cho et al.,2006), and CaRMA1H1 (Lee et al., 2009; Seo et al., 2012). Constitutiveexpression of CaPUB1 and CaRMA1H1 in Arabidopsis resulted in reducedand increased tolerance to dehydration stress, respectively, suggestingthat they are involved in the drought stress responses as negative orpositive factors, respectively (Cho et al., 2006; Lee et al., 2009).

Partial CaDSR6 (C. annuum Drought Stress Responsive 6) cDNA waspreviously identified as a drought-induced gene in hot pepper root tis-sue (Hong and Kim, 2005). However, its cellular roles in terms ofdrought stress tolerance were not yet known. In this report, full-length CaDSR6 cDNA was isolated, and transgenic Arabidopsis plants

147E.Y. Kim et al. / Gene 552 (2014) 146–154

overexpressing CaDSR6 (35S:CaDSR6-sGFP) were constructed. Pheno-typic analysis indicated that the 35S:CaDSR6-sGFP transgenic lineswere more tolerant to high salinity and drought stress than wild typeplants. These results suggested that CaDSR6 plays a role in droughtand salt stress responses as a positive factor.

2. Materials and methods

2.1. Plant materials

Hot pepper (C. annuum cv. Pukang) and Arabidopsis thaliana ecotypeColumbia (Col-0) seeds were soaked in 30% sodium hypochlorite solu-tion (bleach) for 6min and rinsedfive timeswith sterilizedwater. Seed-lings were grown in a 0.5× Murashige-Skoog (MS) medium, includingvitamins, 1% sucrose, and 0.7% phytoagar (pH 5.7), or in soil (SunshineMix 5; Sun Gro, USA) in a 22 °C growth chamber under continuouslight conditions.

2.2. Isolation of a full-length CaDSR6 cDNA clone

The full-length CaDSR6 cDNA clonewas PCR-amplified from a λ-uni-Zap II cDNA library constructed from water-stressed leaves from hotpepper plants (Kim et al., 2010a). The sense oligonucleotide was de-signed from the vector-specific primers corresponding to the T3 pro-moter (Table 1). The sequences corresponding to the 3 ′ end of thepartial CaDSR6 cDNA (Table 1) were used to generate the antisense oli-gonucleotide. PCR was performed with PrimeSTAR™ HS DNA polymer-ase (Takara, Japan) and consisted of 25 amplification cycles with anannealing temperature of 55 °C for 40 s and an elongation temperatureof 68 °C for 1min in an automatic thermal cycler (Life technology, USA).PCR products were introduced into the pGEM-T Easy vector (Promega,USA). The amino acid sequences of CaDSR6 and its homolog proteinsfrom various plant species were aligned with ClustalW using Mega5software (Ryu et al., 2010).

2.3. Generation of 35S:CaDSR6-sGFP transgenic Arabidopsis plants

The full-length CaDSR6 cDNAwas introduced into the correspondingsites of a modified pENTR vector as described by Ryu et al. (2010). Theresulting 35S:CaDSR6-sGFP fusion genewas transferred to Agrobacteriumtumefaciens strain GV3101 by electroporation. Agrobacterium cellscontaining the 35S:CaDSR6-sGFP construct were transformed into

Table 1Gene specific primer sequences used for cloning of CaDSR6 cDNA, construction of transgenic p

Gene Forward primers

For cloning of CaDSR6 cDNAT3 promoter 5′-ATTAACCCTCACTCaDSR6

For construction of 35S:CaDSR6-GFP transgenic plantsCaDSR6-GFP 5′-GCGAATTCATGGG

For RT-PCRCaDSR6 5′-ATGGGTTCATGTGAtACT8 5′-TACTGATTACCTC

For real-time qRT-PCRCaDSR6 5′-GCTCCTTTGCCACCaLEAL1 5′-AAGCGCCTCCAGGCaACT 5′-GTTGTTGCACCACRD20 5′-TTAGCTCCGGTCARD22 5′-AGGGCTGTTTCCARD26 5′-GAAGGTGAGGCGRD29A 5′-CAGGTGAATCAGRD29B 5′-GCAAGCAGAAGARAB18 5′-ATCGATCAAACTCKIN2 5′-GCAACAGGCGGGABF3 5′-TGGAAAAGCAGAABI5 5′-AACATGCATTGGCGAPDH 5′-TGAAATCAAAAAG

Arabidopsis plants using a floral-dip method (Zhang et al., 2006). To ob-tain independent transgenic lines, the T1 seeds collected from regenerat-ed T0 plants were germinated on a 0.5× MS medium with 25 μg/mlBasta. Homozygous T3 lines were obtained by subsequent self-crossingand used for phenotypic analyses. The expression of the transgene wasconfirmed by DNA gel blot analysis, RT-PCR, and immuno-blottingusing anti-GFP antibody (Clontech, Japan) as described by Lee et al.(2009).

2.4. RT-PCR and real-time qRT-PCR

Total leaf RNA (3 μg)was isolated fromhot pepper and the transgen-ic Arabidopsis plants before and after drought, high salt, and ABA treat-ments as described by Kim et al. (2010a). For RT-PCR analysis, 1 μl ofthe first strand cDNA reaction products and high fidelity Ex-Taq poly-merase (Takara, Japan)were used in a total volumeof 30 μl. The reactionconsisted of 27 cycles of 30 s at 95 °C, 40 s at 55 °C, and 1 min at 72 °C.Real-time qRT-PCR was performed in a 96-well plate using an IQ5 lightcycler (Bio-Rad, USA)with SYBR Premix Ex Taq II (Takara, Japan) (Xianget al., 2013). The datawere analyzed using theGenesMacro IQ5 conver-sion template and Genex software (Bio-Rad, USA). The amplification ofthe glyceraldehyde-3-phosphate dehydrogenase C subunit was used asan internal control to normalize the data as described by Kim et al.(2010a).

2.5. Phenotype analysis of wild type and 35S:CaDSR6-sGFP transgenicArabidopsis plants after high salinity treatment

For high salinity treatment,wild type and T3 35S:CaDSR6-sGFP trans-genic (independent lines #1, #2, and #3) seeds were germinated for5 days under 0.5× MS with 1% sucrose media in the presence of differ-ent concentrations (0, 75, 150, and 225 mM) of NaCl and germinationprofiles were observed with regard to cotyledon opening after 5 days.These germinated seedlingswere further grown for 8 dayswith orwith-out NaCl and the growth of roots was monitored with the image-analyzing program SCIONIMAGE (Scion Corp., USA).

Wild type and T3 35S:CaDSR6-sGFP transgenic (lines #1, #2, and #3)seedlings were grown for three days under 0.5× MS with 1% sucrosemedia and then transferred into a medium supplemented with150mMNaCl.Morphological changes, including root length and chloro-phyll content, were determined after 10 days.

lants, RT-PCR and real-time qRT-PCR.

Reverse primers

AAAGGGA-3′5′-CCGACGCTATTTGCAGGAC-3′

TTCATGTGCTTCAGTG-3′ 5′-GC GAATTCCCGACGCTATTTGCAGGAC-3′

CTTCAGTG-3′ 5′-CCGACGCTATTTGCAGGAC -3′ATGAAGATCCTTAC-3′ 5′-AAACGATGTCTCTTTAGTTTAGAAGC-3′

AGTCTTC-3′ 5′-TTCCTACAGGGGTGCTCTTG-3′GTAATAG-3′ 5′-GGTGATGTTGCCAATGACTG-3′CTGAGAG-3′ 5′-CCACATCTGCAAAGCCAGTA-3′CCAGTCA-3′ 5′-CATGTATGGTTTTGGTAATGTTTCC-3′CTGAGG-3′ 5′-CACCACAGATTTATCGTCAGACA-3′GAGAGTG-3′ 5′-CCCGAAACTCTGAGTCAACCT-3′GAGTTGTT-3′ 5′-CCGGAAATTTATCCTCTTCT-3′ACCAATCA-3′ 5′-CTTTGGATGCTCCCTTCTCA-3′ATCAAAGTCTAA-3′ 5′-CGAGCTAGAGCTGGATCCAGA-3′AAAGAGTAT-3′ 5′-CCGGTCTTGTCCTTCACGAA-3′AAAATCAGC-3′ 5′-CAAGCATTGCCTTTTGCAT-3′GGAGT-3′ 5′-TTGTGCCCTTGACTTCAAACT-3′CTATCAAGG-3′ 5′-CATCATCCTCGGTGTATCCAA-3′

148 E.Y. Kim et al. / Gene 552 (2014) 146–154

To examine the effects of high salinity on mature leaves, leaf discsfrom wild type and T3 35S:CsDSR6-sGFP transgenic (lines #1, #2, and#3) plants were floated with various concentrations (0, 150, and300mM) of NaCl for 4 days and their chlorophyll contentwasmeasuredas described by Seo et al. (2012).

2.6. Phenotype analysis of wild type and transgenic Arabidopsis plants afterdrought stress

For soil dehydration treatments, three-week-old wild type and 35S:CaDSR6-sGFP transgenic plants were subjected to drought stress for sixdays by withholding water, after which plants were re-watered forthree days (Cho et al., 2011). Their survival ratios were estimated asre-growth ability when they were returned to normal conditions afterwater stress. A cut rosette water loss (CRWL) assay was performed bythe method described by Kim et al. (2010a).

2.7. Measurement of chlorophyll content

Total chlorophyll (chlorophyll a + b) was extracted from salt anddrought stresses treated wild type and T3 35S:CaDSR6-sGFP transgenicleaves using 80% acetone and analyzed as described previously (Kimet al., 2010a). The content of chlorophyll a + b was shown as mg/g offresh weight (FW) tissue.

3. Results

3.1. CaDSR6 sequence analysis

Based on theDD-PCR and subtractive hybridization analyses, diversesets of partial cDNA clones rapidly induced by dehydration were identi-fied from hot pepper seedlings (Park et al., 2003; Hong and Kim, 2005).One of the isolated clones, CaDSR6, encoded a partial polypeptide withunknown function (Hong and Kim, 2005). The full-length CaDSR6(GenBank accession No. KJ858682) was 1017-bp in length and com-posed of an 85-bp 5′-untranslated region, a 705-bp coding regionencoding 235 amino acids, and a 228-bp 3′-untranslated region(Fig. 1A). The molecular mass of the CaDSR6 protein was found to be25.8 kDa with a calculated pI of 9.7. CaDSR6 contains an Asp-rich do-main in its central region, which is approximately 30 amino acids inlength. A database search revealed that there are CaDSR6 homologsin different dicot plants, including Symphytum tuberosum (potato),Solanum lycopersicum (tomato), Vitis vinifera (grape), Ricinus communis(castor bean), Glycine max (soybean), and A. thaliana (Arabidopsis),as well as in monocot plants, such as Oryza sativa (rice) and Zea mays(maize) (Fig. 1B). Amino acid sequence identities between CaDSR6and dicot homologs were 37–80%. CaDSR6 was most closely relatedto the potatoDSR6 homologwith 80% identity,whereas itwas 37% iden-tical to the Arabidopsis homolog (Fig. 1C). On the other hand, CaDSR6was 33% and 37% identical to monocot rice and maize homologs,respectively (Fig. 1C). The Asp-rich central domain is highly conservedin all DSR6 homologs identified. Cellular functions of DSR6 homologsfrom various plant species with regard to stress-tolerance wereunknown.

The real-time qRT-PCR results presented in Fig. 1D revealed that thesteady state level of CaDSR6 mRNA was enhanced in response todrought and high salt concentration and also by the stress hormoneabscisic acid (ABA) treatment in light-grown two-week-old hot pepperplants. Induction patterns of CaDSR6 were comparable to those ofCaLEAL1. The CaLEAL1 gene was previously shown to be induced by abroad spectrum of abiotic stress in hot pepper plants (Park et al.,2003). These results raised the possibility that CaDSR6 is involved inthe defensive responses to abiotic stress.

3.2. Construction of CaDSR6-sGFP-overexpressing transgenic Arabidopsisplants

Although CaDSR6 is a drought- and high salinity-induced gene(Fig. 1D), there is no information about possible functions of DSR6 ho-mologs in terms of drought stress tolerance. To investigate the cellularrole of CaDSR6, transgenic Arabidopsis plants (35S:CaDSR6-sGFP) thatconstitutively expressed CaDSR6-sGFP under the control of the CaMV35S constitutive promoter were constructed using the Agrobacterium-mediated floral-dip transformation method (Zhang et al., 2006)(Fig. 2A). A number of independent transgenic lines were identifiedbased on Basta resistance and also by genomic Southern blot analysis(Fig. 2B). RT-PCR showed that transgenic progeny contained markedlyenhanced amounts of CaDSR6-sGFPmRNA in normal growth conditions(Fig. 2C). Immuno-blot analysis using anti-GFP antibody indicated thatCaDSR6-sGFP fusion protein was effectively expressed in transgeniclines (Fig. 2D). These CaDSR6-sGFP-overexpressing T3 transgenic lines(#1, #2, and #3) were used for phenotypic analysis.

3.3. Overexpression of CaDSR6 increased tolerance to high salinity treat-ment in Arabidopsis

We first compared the capacity of the 35S:CaDSR6-sGFP transgenicand wild type plants to respond to high salinity treatment ingermination and post-germination stages. Wild type and CaDSR6-overexpressing T3 transgenic (independent lines #1, #2, and #3)seeds were germinated with various concentrations (0, 75, 150, and225 mM) of NaCl, and germination profiles were observed with regardto cotyledon opening after 5 days. The results showed that wild typeand 35S:CaDSR6-sGFP seeds exhibited undistinguishable cotyledonopening under all NaCl concentrations examined (SupplementaryFig. S1), indicating that wild type and CaDSR6-overexpressing seedshave similar germination rates under high salinity conditions.

These germinated seedlings were further grown for 8 days with orwithout NaCl and the growth of roots was monitored. In the presenceof NaCl, wild type seedlings exhibited more retarded root growth com-pared to the 35S:CaDSR6-sGFP seedlings.With 75mMNaCl, roots ofwildtype seedlings were 23.0 ± 0.9 mm long, while those of CaDSR6-overexpressing seedlings were 34.9 ± 1.9 to 39.0 ± 2.4 mm long(Fig. 3A). Differences in root growth between the wild type and trans-genic plantswere still apparent at 150mMof NaCl.Wild type and trans-genic roots were 7.30 ± 1.0 mm long and 11.5 ± 2.0–12.5 ± 2.11 mmlong, respectively. These results indicate that roots of CaDSR6-overexpressing plants were more tolerant to high salinity than werethe wild type roots.

As a next experiment, wild type and 35S:CaDSR6-sGFP seedlings(lines #1, #2, and #3) were grown for three days under normalgrowth conditions and then transferred to a medium supplementedwith 150 mM NaCl. Their morphological changes were monitoredafter 10 days. After 10 days of salt treatment, root lengths fromwild type seedlings were 19.3 ± 2.3 mm, whereas those from 35S:CaDSR6-sGFP seedlings were 30.2 ± 1.6–31.5 ± 2.2 mm (Fig. 3B),which was consistent with the results in Fig. 3A. In addition, wildtype leaves exhibited a pale green phenotype, and their averagechlorophyll content was 2.2 ± 0.4 mg/g FW after salt stress (Fig. 3B).However, CaDSR6-sGFP-overexpressing transgenic leaves appearedgreen and healthy with average chlorophyll levels of 5.4 ± 0.3 to5.9 ± 0.4 mg/g FW.

To examine the effects of high salinity on mature leaves, leaf discsprepared from wild type and T3 35S:CaDSR6-sGFP transgenic (lines #1,#2, and #3) plants were floated with various concentrations (0, 150,and 300 mM) of NaCl for 4 days and then their chlorophyll contentwas measured. Under high salt concentrations, wild type leaves werehighly bleached, whereas the transgenic leaves retained a greenishcolor under high salt conditions (Fig. 3C). After 150mMNaCl treatment,approximately 72.2% and 73.1–79.6% of the total chlorophyll content

A

B

C

397681

HindIII HindIII NdeI SpeI

1017 bp

CaDSR6

S.tubersum

S.lycopersicum

V.vinifera

R.communis

A.thaliana

G.max

Z.mays

O.sativa0.1

D

Fig. 1. Sequence analysis of hot pepper CaDSR6 and its homologs. Schematic representation and restriction enzyme map analysis of the full-length CaDSR6 cDNA clone. Solid lines depictthe 5′- and 3′-untranslated regions. The solid bar indicates the coding region. The dark bar represents a conserved Asp-rich domain whose cellular function is unknown. The sequence ofCaDSR6 has been deposited in the GenBank database under accession number KJ858682 (A); Comparison of the derived amino acid sequence of hot pepper CaDSR6with those of the dicotplants (potato XP_006338688.1, tomato XP_004231788.1, grape XP_002262897.2, castor bean XP_002509538.1, soybean XP_003553921.1, and Arabidopsis NP_186835) and monocotplants (rice NP_001045217 andmaize ACG33331.1). Amino acid residues conserved in all nine proteins are shown in black, and amino acids identical in at least five of the nine sequencesare in gray. A black box indicated the Asp-richmotif among theDSR6 homologs. Dashes indicate gaps in the amino acid sequences for optimal alignment (B); Phylogenetic relationships ofDSR6 homologs fromhot pepper, potato, tomato, grape, castor bean, soybean,Arabidopsis, rice, andmaize. The treewas generated usingMEGA5 softwarewith the neighbor-joiningmeth-od. The tree is drawn to scalewith branch lengths in the sameunits as those of the genetic distances. The 0.1 scale bar implies a genetic distance of 0.1 substitutionsper site (C). Induction ofCaDSR6 in hot pepper leaves in response to dehydration, high salinity, and ABA treatments. Light-grown 2-week-old hot pepper plants were subjected to dehydration stress by exposingwhole plants to a stream of air in a clean bench for 1 h, 300mMNaCl for 3 h, and 100 μMABA for 3 h. Total RNA was isolated from leaves and analyzed by real-time qRT-PCR using gene-specific primers designed for CaDSR6 and CaLEAL1 (a positive control). Themean value of three technical replicates was normalized to the levels of glyceraldehyde-3-phosphate dehydro-genase C subunit mRNA as an internal control (D).

149E.Y. Kim et al. / Gene 552 (2014) 146–154

were retained in thewild type and transgenic leaves, respectively. Wildtype leaves lost 69.0% of their chlorophyll after 300mMNaCl treatment.In contrast, CaDSR6-overexpressing leaves still contained 43.0–65.5% ofthe total chlorophyll content after 300 mM NaCl treatment. Therefore,35S:CaDSR6 seedlings displayed less evident phenotypic anomalies rel-ative to wild type seedlings in response to salt stress, suggesting thatoverexpression of CaDSR6 resulted in increased tolerance to high salin-ity in post-germination growth.

3.4. 35S:CaDSR6-sGFP plants were more tolerant to a water deficit thanwild type plants

Because expression of CaDSR6 was induced by dehydration (Hongand Kim, 2005) and 35S:CaDSR6-sGFP transgenic plants were more tol-erant to high salinity (Fig. 3), we speculated that overexpression ofCaDSR6 also alters the drought stress responses. Thus, wild type andCaDSR6-overexpressing plants were subjected to water stress. These

A

CaDSR6

AtACT8

35S:CaDSR6-sGFP

WT #1 #2 #3

C

3.0

3.5

10.0

8.0

6.0

5.0

4.0

WT #1 #2 #3

35S:CaDSR6-sGFP

Probe : sGFP gene

D35S:CaDSR6-sGFP

WT #1 #2 #3

Anti-GFP

B

70

50

kDa

RbcL

CaDSR6-sGFP

kb

Fig. 2. Construction of CaDSR6-overexpressing transgenic Arabidopsis plants. Schematic structure of the CaDSR6-sGFP overexpression binary vector construct. LB, left border; tMAS,mannopine synthetase terminator; pMAS, mannopine synthetase promoter; p35S, cauliflower mosaic virus 35S promoter; sGFP, soluble green fluorescence protein; tOCS, octopine syn-thase terminator; RB, right border (A); DNA gel blot analysis. Leaf genomic DNA was isolated from wild type (WT) and 35S:CaDSR6-sGFP transgenic (#1, #2, and #3) Arabidopsis plants.DNAwas digested byXbaI and hybridizedwith a 32P-labeled sGFPprobe under high stringency conditions (B); expression levels of CaDSR6mRNAs inWT and transgenicArabidopsis plants.Total leaf RNA was isolated from WT and three independent T3 35S:CsDSR6-sGFP transgenic lines and analyzed by RT-PCR using the gene-specific primers listed in Table 1. AtACT8 is aloading control (C); protein gel blot analysis. Total leaf protein was extracted fromWT and three independent T3 35S:CsDSR6-sGFP transgenic lines, separated by SDS-PAGE, and then sub-jected to immuno-blotting using an anti-GFP antibody (D).

150 E.Y. Kim et al. / Gene 552 (2014) 146–154

plants were grown for three weeks under the normal water conditionsand then grown for six days without irrigation. The survival rates ofdehydrated plants were determined three days after re-irrigation. Theresults revealed that 6.7% (6 of 90) of wild type plants survived and re-sumed their growth after drought-irrigation treatment (Fig. 4A). In con-trast, 35S:CaDSR6-sGFP plants appeared to be more tolerant to waterstress than wild type plants. As demonstrated in Fig. 4A, survival ratesof transgenic lines #1, #2, and #3 were 29.7% (27 of 91), 60.1% (60 of99), and 26.7% (27 of 101), respectively. Thus, constitutive expressionCaDSR6 conferred amarkedly enhanced tolerance to dehydration stress.

Next, the cut rosette water loss (CRWL) rates (Bouchabke et al.,2008) of wild type and 35S:CaDSR6-sGFP plants were determined. De-tached rosette leaves from three-week-old wild type and transgenicplants were incubated on open-lid Petri dishes for 0–5 h at room tem-perature under dim light, and decreases in their fresh weights were re-corded. As shown in Fig. 4B, the fresh weight of detached wild typeleaves was more rapidly reduced than those of 35S:CaDSR6-sGFP plants(lines #1, #2, and #3) during incubation. At 0.5 h of incubation, signifi-cant differences in CRWL rates between wild type and CaDSR6-overexpressing leaves were already evident. At 5 h incubation, thefresh weight of wild type leaves decreased to approximately 26% ofthe starting weight. However, the fresh weight of the transgenic leaveswas reduced to only 39–47% at 5 h incubation depending on the inde-pendent lines. These results indicate that CaDSR6 leaves hold their leafwater more efficiently than wild type leaves.

Leaf chlorophyll content in wild type and CaDSR6-overexpressorswas consistently different after water stress.Wild type leaves contained

13.9 ± 1.0 mg/g FW of total chlorophyll upon drought treatment(Fig. 4C). Under the identical drought conditions, chlorophyll contentin 35S:CaDSR6-sGFP leaves was 17.4 ± 0.9–18.0 ± 0.4 mg/g FW de-pending on the independent transgenic lines (#1, #2, and #3). Collec-tively, phenotypic analysis suggested that 35S:CaDSR6-sGFP plantswere more tolerant to dehydration stress as compared to wild typeplants.

3.5. Drought-induced marker genes were up-regulated in 35S:CaDSR6-sGFP transgenic plants

To examine whether the drought-tolerant phenotype of 35S:CaDSR6-sGFP plants was correlated with changes in the expression ofother stress-related genes, levels of drought-induced marker genes, in-cluding RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5(Lopez-Molina et al., 2001; Finkelstein et al., 2002; Brocard-Gifford etal., 2004; Yamaguchi-Shinozaki and Shinozaki, 2006; Fujita et al.,2009; Kimet al., 2010b; Yoshida et al., 2010; Tao and Lu, 2013),were de-termined by real-time qRT-PCR. The results in Fig. 5 show that all of thestress-induced genes examined were up-regulated in 35S:CaDSR6-sGFPlines (#1 and #2) relative towild type plants under normal growth con-ditions. After drought treatment, the higher levels of drought-responsive transcripts accumulated in 35S:CaDSR6-sGFP plants com-pared to wild type plants. These results indicated that overexpressionCaDSR6 resulted in the increased expression of drought-induced genesin both normal and drought conditions. Overall, the overexpression ofCaDSR6 is linked to increased levels of stress-induced genes, which, in

A

B C35S:CaDSR6-sGFP 35S:CaDSR6-sGFP

WT #1 #2 #3

35S:CaDSR6-sGFP

WT #1 #2 #3

35S:CaDSR6-sGFP

0 mM NaCl 75 mM NaCl

150 mM NaCl

225 mM NaCl

0 mM NaCl 150 mM NaCl

Fig. 3. Tolerant phenotype of 35S:CaDSR6-sGFP transgenic plants in response to high salinity in post-germination stage. Wild type (WT) and CaDSR6-overexpressing T3 transgenic(independent lines #1, #2, and #3) seedswere germinated in the presence of various concentrations (0, 75, 150, and 225mM) of NaCl for 5 days. These germinated seedlingswere furthergrown for 8 days with or without NaCl and the growth of roots was monitored. Error bars indicate mean ± SD (n = 40) ** and * indicate the statistical significance as determined byStudent's t test at P b 0.001 and P b 0.01, respectively. (A). Wild type (WT) and T3 35S:CsDSR6-sGFP transgenic (lines #1, #2, and #3) seedlings were grown for three days under normalgrowth conditions and then transferred to a medium supplemented with 150 mMNaCl. Root growth profiles and amount (mg/g FW) of total chlorophyll (chlorophyll a + b) in leaves ofWT and 35S:CaDSR6-sGFP transgenic seedlingswere determined after salt treatment. Error bars indicate mean± SD (n= 38) ** and * indicate the statistical significance as determined byStudent's t test at P b 0.001 and P b 0.01, respectively. (B). Leaf discs prepared frommature wild type (WT) and 35S:CaDSR6-sGFP transgenic (lines #1, #2, and #3) leaveswere incubatedwith 0, 150, and 300mMNaCl and their relative total chlorophyll content (%) was analyzed after 4 days. Error bars indicatemean± SD (n=9) ** and * indicate the statistical significanceas determined by Student's t test at P b 0.01 and P b 0.05, respectively. (C).

151E.Y. Kim et al. / Gene 552 (2014) 146–154

turn, conferred a drought tolerant phenotype in transgenic Arabidopsisplants.

4. Discussion

The partial CaDSR6 gene was previously identified as a drought-induced gene by the subtractive hybridization method in hot pepperplants (Hong and Kim, 2005). Although CaDSR6 homologs are present

in a wide range of plant species, including Arabidopsis and rice (Fig. 1),their cellular roles have not been addressed. In this report, CaDSR6-overexpressing transgenic Arabidopsis plants were generated (Fig. 2)and their salt and drought stress tolerance were investigated. The re-sults indicated that CaDSR6-overexpressors were highly tolerant toboth high salinity and dehydration treatments in post-germinationstage (Figs. 3 and 4). After salt stress treatment, transgenic seedlings ex-hibitedmore vigorous root growth thanwild type seedlings (Fig. 3A and

A

WT

35S:CaDSR6-sGFP

#1 #2

6 da

ys w

/o

wat

erin

g3

days

afte

r re

-wat

erin

g3-

wee

k-ol

dpl

ants

Drought

Re-watering

#3

6.67%(6/90)

29.67%(27/91)

60.61%(60/99)

26.73%(27/101)

B

C

35S:CaDSR6-sGFP

Fig. 4. Tolerant phenotype of 35S:CaDSR6-sGFP transgenic plants in response to dehydration stress. Wild type (WT) and three independent T3 35S:CaDSR6-sGFP transgenic (lines #1, #2,and#3)plantswere grown for threeweeks under normalwatering conditions and then grown for six dayswithout irrigation. Survival rates of dehydratedplantswere determined at threedays after re-irrigation (A); cut rosette water loss (CRWL) assay. The detached rosette leaves from three-week-oldWT and 35S:CaDSR6-sGFP transgenic (lines #1, #2, and #3) plants wereincubatedonopen-lid Petri dishes for 0–5 h at roomtemperature. Reduction of freshweight (FW)wasdetermined as the percentage of initial FWof thedetached leaves. Error bars indicatemean ± SD (n = 9) (B); amount (mg/g FW) of total chlorophyll (chlorophyll a + b) in leaves of WT and 35S:CaDSR6-sGFP transgenic plants after drought stress. Error bars indicatemean ± SD (n = 9) ** and * indicate the statistical significance as determined by Student's t test at P b 0.01 and P b 0.05, respectively. (C).

152 E.Y. Kim et al. / Gene 552 (2014) 146–154

B). Transgenic leaves remained green in response to high salinity, sug-gesting their higher photosynthetic capacity than wild type leaves(Fig. 3B and C). Mature transgenic plants weremore tolerant to droughtcompared to wild type plants (Fig. 4A). Consistently, 35S:CaDSR6-sGFPleaves retained their water content and chlorophyll more efficientlythan wild type leaves in response to dehydration stress (Fig. 4B andC). Based on these phenotypic analyses, we concluded that constitutiveexpression of CaDSR6 conferred tolerance to salt and drought stresses inArabidopsis.

Expression levels of nine drought-induced marker genes, includingRD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, weremarkedly increased in CaDSR6-overexpressors relative to wild typeplants before and after drought treatment (Fig. 5). These stress-responsive genes are also known to be activated by high salt conditions.Therefore, it is possible that overexpression of CaDSR6 is associated di-rectly or indirectly with the up-regulation of osmotic stress-inducedgenes in both normal and salt and drought stress conditions. One possi-bility is that CaDSR6 is a transcription factor. Thus, overexpression ofCaDSR6 could trigger the constitutive expression of stress genes,which, in turn, resulted in increased salt and drought tolerance in trans-genic Arabidopsis plants. Amino acid sequence analysis of CaDSR6 andits homologs, however, did not indicate any possible nuclear localiza-tion signal or a putative DNA binding motif (Fig. 1). Indeed, subcellular

localization of CaDSR6 was predicted to be cytosolic by two differentprograms (www.cbs.dtu.dk/services/TargetP and http://www.psort.org). Therefore, it is plausible that ectopic expression of CaDSR6 maycause the induction of stress genes by an as-yet unknown indirectmechanism. In this context, it isworthnoting that CaDSR6 and its homo-logs contain a highly conserved domain in their central regions, which isapproximately 30 amino acids in length. This short domain is rich in Aspamino acid and CaDSR6 possesses six Asp/Glu residues, including anAsp–Asp–Asp tri-peptide sequence. At thismoment, however, function-al relevance of the Asp-rich domain of CaDSR6 in drought tolerance re-mains to be solved.

Interestingly, the Asp-rich regions of DSR6 proteins display structur-al similarity to the N-terminal region of yeast p23fyp and human hTCTPproteins (Fig. 6). Yeast p23fyp and human hTCTP belong to a structuralsuperfamily of Mss4/Dss4 proteins, which interact with the GDP/GTPfree form of Rab proteins (Thaw et al., 2001; Feng et al., 2007). Mss4 isknown to be a guanine nucleotide-free chaperone, and it also acts as aguanine nucleotide exchange factor (Boguski and McCormick, 1993;Pai, 1998). This suggested that yeast p23fyp and human hTCTP haveRab protein binding sites (Thaw et al., 2001). The N-terminal region ofapproximately 30 amino acids in Mss4/Dss4 superfamily proteins, in-cluding p23fyp and hTCTP, forms five short β-sheet structures (Fig. 6).Similarly, the Asp-rich region of DSR6 homologs was predicted to

before drought after drought

before drought after drought

before drought

after drought before drought after drought before drought

after drought before drought after drought before drought

after drought before drought after drought before drought after drought

Fig. 5. Real-time qRT-PCR analysis of drought-induced genes in wild type and 35S:CaDSR6-sGFP transgenic plants. Total RNA was obtained from wild type (WT) and T3 35S:CsDSR6-sGFPtransgenic (lines#1 and#2)plants before and after drought treatment and analyzed by real-time qRT-PCRusing the gene-specific primers listed in Table 1. The graphs indicate the relativeexpression levels of the RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5 genes inWT and CaDSR6-overexpressors. Themean value of three technical replicates was normal-ized to the levels of glyceraldehyde-3-phosphate dehydrogenase C subunit mRNA as an internal control.

p23fyp

hTCTP

CaDSR6S.tuberosumS.lycopersicumV.viniferaR.communis

44

6164646350

3637

9598989684

Fig. 6. Amino acid sequence alignment of the N-terminal region of yeast p23fyp andhuman hTCTP proteins and the Asp-rich region of DSR6 homologs. The predicted five sec-ondary β-sheet structures are indicated with arrows. Ser, Glu, and two Asp residuesconserved in all seven proteins from yeast, human, and plants are shown in black.Amino acid residues identical in at least four of the seven sequences are shaded. Thedark circle indicates the Glu amino acid residue that is essential for the interaction withRab proteins.

153E.Y. Kim et al. / Gene 552 (2014) 146–154

form very similar five β-sheet structures. Amino acid sequence align-ment showed that Ser, Glu, and two Asp residues are well conservedin the domains of DSR6 homologs and in yeast and human proteins(Fig. 6). The conserved Glu residue is an essential amino acid for the in-teraction with Rab proteins (Thaw et al., 2001).

In halophyte Mesembryanthemum crystallinum, RabF1 gene expres-sionwas enhanced in response to high salinity (Bolte et al., 2000). In ad-dition, ectopic expression of Arabidopsis RabG3e (AtRab7) resulted inincreased tolerance to osmotic stress induced by NaCl and sorbitol(Mazel et al., 2004). These results suggest that Rab proteins are involvedin plant adaptation to salt and osmotic stresses. With this in mind, weconsidered the possibility that CaDSR6 may play a positive role indrought and salt stress responses through interaction with the Rabfamily, and that the conserved Asp-rich domain may be involved inthe interaction between CaDSR6 and Rab proteins. A more detailedfunctional characterization of CaDSR6 will be required to test thishypothesis.

154 E.Y. Kim et al. / Gene 552 (2014) 146–154

In conclusion, 35S:CaDSR6-sGFP transgenic Arabidopsis plants werefound to be more tolerant to high salinity and drought stress thanwild type plants, suggesting a critical role of hot pepper CaDSR6 in re-sponse to drought and salt stresses as a positive factor.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2014.09.028.

Conflict of interest

Authors declare that that is no conflict of interest.

Acknowledgments

Thisworkwas supported by a grant from theWoo Jang Chun SpecialProject (PJ009106) funded by the Rural Development Administration,Republic of Korea, to W.T.K.

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