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Plant Mol Biol (2014) 86:609–625DOI 10.1007/s11103-014-0251-4

Arabidopsis drought‑induced protein Di19‑3 participates in plant response to drought and high salinity stresses

Li‑Xia Qin · Yang Li · Deng‑Di Li · Wen‑Liang Xu · Yong Zheng · Xue‑Bao Li

Received: 7 January 2014 / Accepted: 5 September 2014 / Published online: 14 September 2014 © Springer Science+Business Media Dordrecht 2014

exhibited more drought-sensitivity than wild type. Fur-thermore, expression of the genes related to ABA signal-ing pathway was altered in Atdi19-3 mutant and AtDi19-3 transgenic plants. These data suggest that AtDi19-3 may participate in plant response to drought and salt stresses in an ABA-dependent manner.

Keywords Arabidopsis thaliana · Drought-induced (Di19) protein · Abscisic acid (ABA) · Drought/high salinity stress · Seedling development

Introduction

Drought, high salinity and low temperature are the most common abiotic stresses that limit distribution of plants and affect crop productivity and quality (Xiong et al. 2002; Jakab et al. 2005). To respond and adapt to these stresses, plants have developed a complex of molecular, biochemical and physiological mechanisms by modulating the expres-sion of specific sets of genes (Shinozaki et al. 2003). Sev-eral signal transduction pathways exist in plants responding to abiotic stresses, including calcium-independent mito-gen-activated protein kinase (MAPK) cascade signaling, calcium-dependent protein kinases (CDPK) phosphoryl-ated signal pathway, calcium-dependent SOS (salt overly sensitive) pathway and others (review by Xiong et al. 2002). For instance, expression of Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize (Shou et al. 2004). Overexpression of AtCPK4 or AtCPK11 in Arabidopsis enhanced ABA/salt sensitivity in seed ger-mination and seedling growth, but loss-of-function muta-tions of CPK4 and CPK11 resulted in ABA/salt insensitive phenotypes (Zhu et al. 2007). Arabidopsis sos3 mutant is hypersensitive to Na+ and Li+ stresses, and external Ca2+

Abstract Di19 (drought-induced protein19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, Arabidopsis Di19-3 was functionally characterized. The experimental results revealed that AtDi19-3 is a tran-scriptional activator, and could bind to the TACA(A/G)T sequence. AtDi19-3 expression in plants was remark-ably induced by NaCl, mannitol and abscisic acid (ABA). T-DNA insertion mutation of AtDi19-3 results in an increase in plant tolerance to drought and high salinity stresses and ABA, whereas overexpression of AtDi19-3 leads to a drought-, salt- and ABA-sensitive phenotype of the transgenic plants. In the presence of NaCl, mannitol or ABA, rates of seed germination and cotyledon green-ing in Atdi19-3 mutant were higher, but in AtDi19-3 over-expression transgenic plants were lower than those in wild type. Roots of Atdi19-3 mutant seedlings were longer, but those of AtDi19-3 overexpression transgenic seedlings were shorter than those of wild type. Chlorophyll and proline contents in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression seedlings were lower than those in wild type. Atdi19-3 mutant showed greater drought-tol-erance, whereas AtDi19-3 overexpression transgenic plants

Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0251-4) contains supplementary material, which is available to authorized users.

L.-X. Qin · Y. Li · D.-D. Li · W.-L. Xu · Y. Zheng · X.-B. Li (*) Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan 430079, Chinae-mail: xbli@mail.ccnu.edu.cn

Present Address: L.-X. Qin Institute of Cotton, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, China

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can improve its potassium nutrition and salt tolerance (Liu and Zhu 1997; Guo et al. 2001).

ABA is required for plant adaptation to environmental stress by affecting some physiological processes in plant development, particularly in seed dormancy and germina-tion, and early seedling development (Belin et al. 2009). In recent years, ABA signal transduction pathway plays a significant role in plant response to drought/salt stress. Some key elements, including both negative and posi-tive regulators, have been identified in plants (Himmel-bach et al. 2003; Israelsson et al. 2006). In ABA-depend-ent pathway, MYC and MYB recognition sequences are essential for the ABA- and drought-responsive expression of rd22 (Abe et al. 1997). The ABA-responsive regula-tory elements (ABREs) and MYC/MYB systems function in the adaptive stress response process after accumulation of endogenous ABA in dehydration conditions (Shinozaki and Yamaguchi-Shinozaki 2000). Some transcription fac-tors (TFs), such as ABA-responsive element binding pro-teins (ABI/ABF/AREB/bZIP families), play positive roles in ABA signaling (Choi et al. 2000; Chak et al. 2000; Uno et al. 2000). In addition, ABA-independent pathway has been proposed to exist in plants for regulating expression of genes (e.g. rd19, rd21, erd1 and erd15) to respond to drought and high salinity (Simpson et al. 2003). Expression of a rab-related gene, RAB18, is induced by ABA during cold acclimation process of Arabidopsis (Lang and Palva 1992), but the rate of RAB18 expression is independent of the level of ABA uptaken by Arabidopsis suspension cells (Jeannette et al. 1999). Overexpression of EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) reduced plant ABA sensitivity and impaired plant drought and freezing tolerance. In contrast, RNAi silencing of ERD15 resulted in plant hypersensitive to ABA and improved plant tolerance to both drought and freezing (Kariola et al. 2006). It has been reported that the same gene may be activated by different pathways in different stresses. For example, the dehydration-responsive expression of RD26 is regulated mainly by ABA, but an ABA-independent pathway of NaCl signaling for RD26 expression is found in the ABA-defi-cient aba2 mutant (Fujita et al. 2004). Moreover, genetic evidence suggested that stress-signaling pathways for the activation of LEA-like genes which are completely inde-pendent of ABA may not exist (Xiong et al. 2002). There-fore, both ABA-dependent and ABA-independent signal transduction pathways may interact and converge to acti-vate stress-response genes.

Cys2/His2-type zinc-finger proteins (ZFPs), also called classical TFIII-types zinc-finger proteins, represent a large family of eukaryotic transcription factors. The Cys2/His2-type zinc finger domain containing two cysteines and two histidines is one of the best-characterized and important

DNA-binding motifs involved in protein-DNA interac-tion in plants (Takatsuji 1999; Pabo et al. 2001). However, subsequently some studies have shown that some Cys2/His2-type zinc-finger motifs can bind to RNA (Searles et al. 2000), and some may participate in protein–protein interaction (Wolfe et al. 2000). Cys2/His2-type zinc-finger domain consists of ~30 amino acid residues, and its con-sensus sequence is CX2–4CX3FX5LX2HX3–5H. In these amino acids, a zinc ion is tetrahedrally coordinated by two cysteines and two histidines in order to maintain its sta-bility (Pabo et al. 2001). In Arabidopsis, 176 zinc-finger proteins that contain one or more zinc-finger motifs have been identified (Englbrecht et al. 2004). Several plant zinc-finger proteins have been found to play important roles in plant response to abiotic stresses. For example, Arabi-dopsis plants overexpressing a zinc finger protein RHL41 displayed an increased tolerance to high light intensity, and also morphological changes of thicker and dark green leaves (Lida et al. 2000). Transgenic Arabidopsis overex-pressing STZ, as a transcription repressor, showed growth retardation and increased tolerance to drought stress (Saka-moto et al. 2004). Soybean SCOF-1 enhanced cold toler-ance of the transgenic plants via protein–protein interaction (Kim et al. 2001). Overexpression of rice ISAP1 in tobacco resulted in the increased tolerance to drought, salt and cold stresses (Mukhopadhyay et al. 2004). ThZF1, a Cys-2/His-2-type transcription factor from salt cress (Thellungiella halophila), is involved in drought and salt stress (Xu et al. 2007). In addition, loss of rice DST (drought and salt tol-erance) protein function resulted in enhanced drought and salt tolerance in rice (Huang et al. 2009).

Di19 (drought-induced 19) proteins contain two unusual Cys2/His2 (C2H2) zinc-finger domains that are evolution-arily well conserved (Gosti et al. 1995). They may share a common or closely related biological function, based on their sharing of a common conserved C2H2 zinc finger-like motif. In Arabidopsis, Di19 family contains seven hydro-philic protein members. Five of seven AtDi19 proteins are preferentially localized to cell nucleus. AtDi19-1 and AtDi19-3 are rapidly induced by dehydration, and transcript amounts of AtDi19-2 and AtDi19-4 increased in response to high-salinity stress. However, most of AtDi19 genes are not transcriptionally induced by ABA. Besides, two cot-ton Di19 proteins, named GhDi19-1 and GhDi19-2, are involved in plant response to salt stress and ABA signal-ing. Overexpression of GhDi19-1 and GhDi19-2 in Arabi-dopsis resulted in the increased sensitivity to high salinity and exogenous ABA (Li et al. 2010). Recently, a study reported that Arabidopsis Di19-1 as a transcription factor participates in response to drought stress by binding to the TACA(A/G)T element within the promoters of PR1 (patho-genesis-related 1), PR2 and PR5 genes (Liu et al. 2013). In

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this study, we report that AtDi19-3 as a transcriptional acti-vator is involved in plant response to high salinity, drought, ABA and H2O2. Atdi19-3 T-DNA insertion mutant shows the enhanced tolerance to salt/osmotic stress and exog-enous ABA, but sensitive to H2O2 during seed germination and early seedling development, while AtDi19-3 overex-pression transgenic plants display the opposite phenotype.

Materials and methods

Plants materials and growth conditions

A T-DNA insertion mutant (named Atdi19-3, Gabi_853B10) of Arabidopsis Di19-3, AT3g05700 was obtained from ABRC (www.arabidopsis.org/abrc). Seeds of Arabidopsis thaliana (Columbia ecotype) were surface-sterilized with 75 % ethanol for 1 min and 10 % NaClO for 3 min, fol-lowed by washing with sterile water. The sterilized Arabi-dopsis seeds were plated on Murashige and Skoog (MS) medium. After stratification at 4 °C for 2 days, the plates were transferred to a plant growth incubator (Sanyo, Osaka, Japan) for seed germination (16 h light/8 h dark at 22 °C) 10 days later, seedlings were transplanted in soil and grown in a growth room under the conditions of 16 h light/8 h dark cycle, 22–24 °C. Tissues were derived from these seedlings for RNA isolation.

Subcellular localization and transcriptional activity analysis

The coding sequence of AtDi19-3 gene was cloned into a pBI121-eGFP vector at Xbal I/BamH I to generate 35S:AtDi19-3-eGFP construct (Fig. S1C), and then intro-duced into Arabidopsis by the floral dip method (Clough and Bent 1998). The harvested seeds were germinated on selective MS medium for selecting transgenic plants. Sub-sequently, fluorescence microscopy was performed on a SP5 Meta confocal laser microscope (Leica, Germany). Roots of the transgenic seedlings were examined with a filter set for GFP fluorescence (488 nm for excitation and 506 ~ 538 nm for emission). SP5 software (Leica, Germany) was employed to record and process the digital images taken. Primers used in AtDi19-3:eGFP vector construc-tion as follows: AtDi19-3 P1 5′-GGGTCTAGAATGGA TTCCGATTCATGGAG-3′ and P2 5′-GGGGGATCCTAA GCTGTCATCAAGAATCG-3′.

The coding sequence of AtDi19-3 gene was cloned into pGBKT7 vector (Biosciences Clontech, Palo Alto, CA, USA) containing GAL4 DNA binding domain (BD) (Fig. S1A). The BD-AtDi19-3 construct was transferred into yeast strains AH109 and Y187, respectively. Three reporter

genes HIS3 (histidine), ADE2 (adenine) and lacZ were tested by streaking the yeast AH109 transformants on SD/-Trp/-His and SD/-Trp/-Ade medium (SD minimal medium lacking Trp and His or lacking Trp and Ade) (Clontech Inc., Palo Alto, CA, USA), and β-galactosidase (β-gal) activity of yeast Y187 transformants was determined by the flash-freezing filter assay. Primers used in AtDi19-3-BD vector construction as follows: AtDi19-3 P1 5′-GGGCATATGATG GATTCCGATTCATGGAG-3′ and P2 5′-GGGGTCGACTT ATAAGCTGTCATCAAGA-3′.

EMSA assay

Electrophoretic mobility shift assay (EMSA) was carried out using a molecular probes’ fluorescence-based EMSA Kit (Invitrogen). The coding sequence of AtDi19-3 was inserted downstream the malE gene, which encodes maltose-bind-ing protein (MBP), in pMAL-c2X vector for expressing MBP-AtDi19-3 fusion protein (Fig S1B). Primers used in pMAL-c2X-AtDi19-3 vector construction as follows: AtDi19-3 P1 5′-CTTGGATCCATGGATTCCGATTCATG GAG-3′ and P2 5′-GGGGTCGACTTATAAGCTGTCATC AAG-3′. The MBP-AtDi19-3 fusion protein purified from Escherichia coli strain BL21 by MBP’s affinity for malt-ose (NEW ENGLAND) was used in protein/DNA binding analysis, using MBP protein as control. A pair of 30 bp oli-gonuleotides (DIBS P1 TACARTTACARTTACARTTACA RTTACART and DIBS P2 AYTGTAAYTGTAAYTGTAA YTGTAAYTGTA) from five short tandem TACA(A/G)T repetitive sequences was synthesized and annealed as DNA probe for EMSA assay. The MBP-AtDi19-3 fusion protein and DNA probe binding reaction was performed in binding buffer (750 mM KCl, 0.5 mM dithiothreitol (DTT), 0.5 mM EDTA, 50 mM Tris–Cl, pH = 7.4) and incubated at room temperature for 20 min. The reaction mixture was separated by non-denaturing polyacrylamide gel electrophoresis. The gel was stained with SYBR® Green EMSA Nucleic Acid Gel Stain and imaged at 254 nm UV epi-illumination.

Construction of AtDi19-3 promoter:GUS chimeric gene and histochemical assay of GUS activity

A 938 bp 5′-flanking fragment of AtDi19-3 gene was cloned into pBI101 vector to generate the chimeric AtDi19-3 promoter:GUS construct (Fig. S1D). The AtDi19-3 promoter:GUS transgenic Arabidopsis was obtained by the floral dip method (Clough and Bent 1998). Histochemical assay of GUS activity in the trans-genic Arabidopsis was conducted according to a modi-fied protocol (Xu et al. 2013). Seedlings (5- and 10-day-old) and mature leaves were collected for assaying GUS expression. The samples were stained at 37 °C 6-8 h in

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5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc) solution. Chlorophyll was cleared from plant tissues by immersing them in 70 % ethanol. To assay the induced GUS expression in plants under salt, ABA and drought treatments, the 10-day-old transgenic seedlings were cul-tured in MS liquid medium containing 150 mM NaCl, 100 μM ABA or 300 mM mannitol for 6 h, and total RNA was extracted from the treated seedlings and con-trols. GUS staining patterns were confirmed by observ-ing at least four different transgenic lines. The repre-sentative stained seedlings or tissues were imaged using a Leica MZ16f stereomicroscope (Leica, Germany). Primers used in the chimeric AtDi19-3 promoter:GUS vector construction as follows: AtDi19-3 promoter P1 5′-GGGAAGCTTAACAGCTCAAATAAACCC-3′ and P2 5′-GGGGGATCCTATTGACAAAACCCGGAAA-3′.

Construction of AtDi19-3 overexpression vector and phenotypic analysis of the transgenic Arabidopsis plants

To construct AtDi19-3 overexpression and complementa-tion mutant vector, the coding sequence of AtDi19-3 gene was cloned into pMD vector under the control of CaMV 35S promoter (Fig. S1F) and into pCAMBIA1301 vector under the control of AtDi19-3 promoter (Fig. S1E), respec-tively. Primers used in the vector construction are: AtDi19-3 P1 5′-CTTGGATCCATGGATTCCGATTCATGGAG-3′ and P2 5′-GGGGTCGACTTATAAGCTGTCATCAAG-3′. The constructs were then transferred into Arabidopsis by the floral dip method (Clough and Bent 1998). Seeds were harvested and stored at 4 °C. Positive transformants were selected on MS medium with 50 mg/L kanamycin or 50 mg/L hygromycin.

AtDi19-3-overexpression and complemented mutant trans-genic Arabidopsis lines were named as 35S:AtDi19-3oe and Atdi19-3 + proAtDi19-3:AtDi19-3oe, respectively. Homozygous plants of T3 and T4 generations were used for phenotypic analysis. Total RNA was extracted from 10-day-old seed-lings of wild type, 35S:AtDi19-3-oe, Atdi19-3 + proAtDi19-

3:AtDi19-3oe and Atdi19-3 mutant. Real-time quantitative RT-PCR (qRT-PCR) analysis was performed as described as previously (Li et al. 2005), using AtDi19-3 gene-specific primers (forward 5′-TCTCTTTCAGCTGAGGATCAC-3′ and reverse 5′-CATGACCTACAAGCAATTGGG-3′).

Seeds of wild type and independent transgenic lines overexpressing AtDi19-3 were germinated on MS medium supplemented with or without 150 mM, 200 mM NaCl, 0.8 and l μM ABA, 300 mM mannitol and 5 mM H2O2, respec-tively (22 °C, 16 h light/8 h dark) in a plant growth incuba-tor. Seeds were considered successfully germinated when radicals completely penetrated the seed coats. Germination

rate and proportion of seedlings with opened green cotyle-dons were expressed as a percentage of the total number of seeds plated.

The seedling growth experiments were performed as described previously (Zhu et al. 2007). Seeds were germi-nated after stratification on MS medium for 48 h and then transferred to MS medium containing 150 mM NaCl, 5 μM ABA, 300 mM mannitol and 5 mM H2O2 in the vertical posi-tion. The status of seedling growth was recorded for 10 days and the length of seedling primary roots was measured at tenth day after the transfer. All statistical analysis experi-ments were performed with three technical replications, each line containing at least 100 seeds for analyzing the seed ger-mination rate and cotyledon greening rate or 30–60 seedlings for vertical cultivation, and repeated at least 3 times.

The chlorophyll content in leaves of 10-day-old seed-lings under 150 mM NaCl, 300 mM mannitol or 5 μM ABA treatment was determined. In brief, chlorophylls in leaves were extracted with 80 % acetone, and chlorophyll content was assayed by measuring absorbance at 645, 652, and 663 nm with a spectrophotometer (Qin et al. 2013). The assays were repeated three times along with three inde-pendent repetitions of the biological experiments.

Proline content in both control and transgenic plants was determined using the protocol as described by Gong et al. (2012). In brief, proline in seedlings was reacted with a mix-ture of 3 % sulphosalicylic acid, glacial acetic acid and 2.5 % ninhydrin in a boiling water bath for 1 h, and then extracted with toluol. Subsequently, proline content was assayed by measuring absorbance at 520 nm with a spectrophotometer. The assays were repeated three times along with three inde-pendent repetitions of the biological experiments.

Drought conditions

Seven-day-old seedlings of wild-type and transgenic plants (approximately 30 of each lines) germinated on MS medium were grown in soil under long-day conditions (16 h light/8 h dark) at 22–24 °C with normal watering for 3 weeks before water was withheld. After water was with-held for 10 days, plants were again watered and photos were taken.

The water loss of detached leaves was measured by weighing the leaves from 3-week-old plants at a specified time (0, 1, 2, 3, 4 and 5 h). Twenty fully expanded leaves were harvested and then weighed (Zhu et al. 2007).

Quantitative RT-PCR analysis

To assay the expression of stress-relative and ABA-respon-sive genes, qRT-PCR analysis was performed with the RNA samples isolated from 10-day-old seedlings treated with or

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without 150 mM NaCl, 300 mM mannitol and 100 μM ABA for 6 h. Total RNA was reversely transcribed into cDNAs, and PCR amplification was performed with oligonucleotides specific for various stress-/ABA-responsive genes (Li et al. 2010): AtABI1 (At4g26080) forward 5′-AGATGGCAAGG AAGCGGATT-3′ and reverse 5′-CAACCACCACCACAC TTATG-3′; ABF4 (At3g19290) forward 5′-AACAACTTAG GAGGTGGTGGTCAT-3′ and reverse 5′-TGTAGCAGCTGG CGCAGAAGTCAT-3′; AtRAB18 (At5g66400) forward 5′-AGATGGCAAGGAAGCGGATT-3′ and reverse 5′-CTTC TTCTCGTGGTGCTCAC-3′; AtERD15 (At2g41430) forward 5′-TCAGCGAGGCTGGTGGATG-3′ and reverse 5′-TGAGA ATGGCGATGGTATCAGGA-3′; AtSOS2 (At5g35410) for-ward 5′-GGCTTGAAGAAAGTGAGTCTCG-3′ and reverse 5′-GCTACATAGTTCGGAGTTCCACA-3′. Expression lev-els of Arabidopsis ACTIN2 were monitored with forward 5′-GAAATCACAGCACTTGCACC-3′ and reverse 5′-AAGC CTTTGATCTTGAGAGC-3′ primers to serve as a normali-zation control.

The expression of these genes was analyzed by quantita-tive RT-PCR using the fluorescent intercalating dye SYBR-Green in a detection system (Opticon2; MJ Research) as described previously (Li et al. 2005). For all the above quan-titative real-time PCR analysis, the assays were repeated three times along with three independent repetitions of the biological experiments, and means of three biological experiments were calculated for estimating gene expression levels.

Results

AtDi19-3 functions as a transcription activator

Sequence analysis showed that AtDi19-3 protein contains a conserved nuclear localization signal region (NLS) next to the two zinc finger domains in its sequence (Milla et al. 2006). To confirm its nuclear localization, AtDi19-3 was fused with an enhanced GFP (eGFP) reporter gene and expressed constitutively under the control of a cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis. As shown in Fig. 1a, GFP fluorescence was strongly accumu-lated mainly in the nuclei of root cells of the transgenic seedlings, demonstrating that AtDi19-3 is a nuclear-local-ized protein.

To analyze the transcription activity of AtDi19-3, an autonomous gene activation test was performed in yeast system. AtDi19-3 was fused to the binding domain (BD) of yeast transcription factor GAL4, and transferred into yeast strain AH109 and Y187 for excluding false posi-tives. The yeast transformants were examined for their growth on selection medium (SD/-Trp-His or SD/-Trp-Ade) based on activation of the HIS3 and ADE2 reporter genes in yeast strain AH109, and the transactivation activity of AtDi19-3 in Y187 strain was determined by β-galactosidase (β-gal) activity due to the activation of the reporter LacZ gene. On minimal synthetic dextrose (SD) medium lacking Trp, yeast strains with both BD and

Fig. 1 Subcellular localization and transcriptional activation analy-sis of AtDi19-3 protein. a Nuclear localization of AtDi19-3 protein. Micrographs of root cells of 35S:AtDi19-3-GFP transgenic Arabi-dopsis. Confocal images were taken under the GFP channel (upper), and with transmitted light (midst), and the upper and midst images were merged (lower). Scale bar 50 μm. b Transcriptional activation assay of AtDi19-3 in yeast. The growth of yeast strain AH109 with

pGBKT7 and pGBKT7-AtDi19-3 constructs under SD/-Trp, SD/-Trp/-Ade and SD/-Trp/-His nutrition-deficient medium. The tran-scriptional activity of AtDi19-3 was measured by β-galactosidase (β-gal) activity assay of yeast Y187 transformants. pGBKT7 is a negative control (−) and pBD-GAL4 is a positive control (+). The pBD-GAL4 and pGBKT7 vectors were used as positive and negative controls, respectively

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BD-AtDi19-3 vectors grew well. On double nutrition-deficient SD medium (SD/-Trp-His or SD/-Trp-Ade), however, only transformants with BD-AtDi19-3 proteins grew well. The β-gal activity assay further confirmed the above results, indicating that AtDi19-3 shows strong tran-scription activation activity (Fig. 1b).

AtDi19-3 protein binds to the TACA(A/G)T element

The DNA-binding sequences of four Cys2His2 zinc-finger proteins were identified in the bacterial one-hybrid system (Meng et al. 2005). Arabidopsis Di19 (AtDi19-1) could bind to the conserved sequence TACA(A/G)T (a novel cis-element, named DIBS) by electrophoretic mobility shift assay (EMSA) (Liu et al. 2013). To investigate whether AtDi19-3 has the ability to binding to the cognate elements, DNA–protein binding assay was carried out by EMSA assay. Five short tandem repetitive sequences (TACA(A/G)TTACA(A/G)TTACA(A/G)TTACA(A/G)TTACA(A/G)T) were used as DNA probe, and AtDi19-3 protein was expressed and purified by pMAL-c2X system. After stained DNA with SYBR® Green EMSA Nucleic Acid Gel Stain, a large molecular weight DNA band was presented in MBP-AtDi19-3/DNA lane, whereas the signals were not detected in MBP/DNA and DNA alone lanes. The results showed that the AtDi19-3 protein could bind to the conserved DIBS element TACA(A/G)T (Fig. 2).

AtDi19-3 promoter is salt-, drought- and ABA-inducible

AtDi19-3 was expressed in seedlings, roots, rosette leaves, flowers and siliques, and AtDi19-3 transcript abundance was higher in rosette leaves and seedlings (Milla et al. 2006). Analysis of a 938 bp AtDi19-3 promoter sequence by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) revealed it contains ABRE and MBS cis-acting elements involved in response to ABA and drought stress. To determine whether AtDi19-3 promoter is induc-ible in plants under salt, drought and ABA treatments, AtDi19-3 promoter:GUS fusion expression vector was con-structed and transferred into Arabidopsis. Histochemical staining of GUS activity revealed that AtDi19-3 promoter was active in cotyledons and roots, especially at the early developmental stages of seedlings (Fig. 3a, c). With fur-ther development, weaker GUS staining was still observed in the mature leaf tips of AtDi19-3 promoter:GUS trans-genic plants (Fig. 3b). We further assayed GUS activity in the transgenic plants under salt, ABA and drought treat-ments. The experimental results revealed that GUS activ-ity was significantly increased in cotyledons of AtDi19-3 promoter:GUS transgenic seedlings (10 day-old) treated with 150 mM NaCl (Fig. 3d), 300 mM mannitol (Fig. 3e)

and 100 μM ABA (Fig. 3f), compared with that of mock treatments (Fig. 3c). A substantial increase in GUS activity was mainly detected in vascular bundle tissues of cotyle-dons and true leaves after salt, ABA and drought treatments (Fig. 3d–f). In addition, we determined expression levels of both GUS and AtDi19-3 genes in the transgenic plants and wild type by quantitative RT-PCR. As shown in Fig. 3g, h, the expression of GUS gene and AtDi19-3 gene in the trans-genic plants with NaCl, mannitol and ABA treatments was remarkably stronger than those without NaCl, mannitol or ABA treatment, and AtDi19-3 transcripts were increased in wild type under NaCl, mannitol and ABA treatments. These results suggested that the AtDi19-3 promoter is salt-, drought- and ABA-inducible.

AtDi19-3 is involved in response to salt, mannitol and ABA during seed germination

To characterize the function of AtDi19-3 gene in plant development, an Arabidopsis Di19-3 (AT3g05700) T-DNA

Fig. 2 DNA-binding assay of AtDi19-3 protein. EMSA assay for AtDi19-3 protein binding DIBS (TACA(A/G)T) sequence (see “Methods”). The gel was stained with SYBR® Green EMSA Stain. The MBP-AtDi19-3/DNA is observed in DNA staining, using MBP protein as negative control

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insertion mutant (Gabi_853B10) (named Atdi19-3) was obtained from Jeff lab and the homozygous mutant line was generated. Atdi19-3 mutant contained a T-DNA insertion in the fourth intron of AtDi19-3 gene (Fig. 4a). Meanwhile, coding sequence of AtDi19-3 was inserted into the respec-tive plant expression vectors pMD driven by CaMV 35S promoter (Fig S1F) and pCAMBIA1301 under the con-trol of AtDi19-3 promoter (Fig. S1E) and introduced into Arabidopsis. Quantitative RT-PCR analysis demonstrated that the transcript levels of the AtDi19-3 were undetect-able in Atdi19-3 T-DNA insertion mutant. Homozygotes of T3 generation of AtDi19-3 overexpression transgenic lines (L4 and L6) and complemented lines (L1 and L5) were analyzed by PCR to ensure the presence of the respective transgene and by quantitative RT-PCR to confirm that the transgene was expressed (Fig. 4b).

Previous reports indicated that AtDi19 genes are tran-scriptionally induced by dehydration, high-salinity stress but not ABA (Milla et al. 2006). To investigate the func-tion of AtDi19-3 gene in plant, seeds of wild type, Atdi19-3 mutant and its complemented lines (L1 and L5, expres-sion of AtDi19-3 driven by itself promoter), and AtDi19-3 overexpression transgenic plants (L4 and L6, driven by

CaMV 35S promoter) were sowed on MS agar medium with or without NaCl, mannitol, ABA and H2O2, respec-tively. Seed germination rates of wild type and AtDi19-3 lines were monitored under different abiotic stresses. All the seeds from wild type and AtDi19-3 lines on MS medium were able to fully germinate (≤4 days) after sowing and showed no significant difference (Fig. 4c). However, in the presence of 150 mM NaCl, seeds from Atdi19-3 mutant germinated much earlier and faster than those of the wild type, whereas AtDi19-3 overexpres-sion transgenic seeds (L4 and L6) germinated much later than wild type (Fig. 4d). After 4 days, about 70 % of wild type seeds germinated, but approximately 85 % of Atdi19-3 mutant seeds germinated, and only about 45 % of AtDi19-3 overexpression transgenic seeds (both L4 and L6) germinated. After 6 days, Atdi19-3 mutant seeds have successfully fully germinated, and germination rate of wild type reached to about 90 %, but AtDi19-3 overex-pression transgenic seeds reached to only approximately 65 % (Fig. 4d). Likewise, in the presence of 300 mM mannitol, seed germination rate of Atdi19-3 mutant was higher than that of wild type, whereas seed germination rate of AtDi19-3 overexpression transgenic lines (L4 and

Fig. 3 Histochemical assay of GUS activity under the control of AtDi19-3 promoter in transgenic Arabidopsis. a A five-day-old seed-ling. b A four-week-old mature leaf. c A ten-day-old seedling. d–f Ten-day-old seedlings treated with 150 mM NaCl (d), 300 mM man-nitol (e) or 100 μM ABA (f). g, h Quantitative RT-PCR analysis of expression of GUS and AtDi19-3 genes in AtDi19-3 promoter:GUS transgenic Arabidopsis plants under NaCl, mannitol and ABA treat-ments, respectively. Ten-day-old AtDi19-3 promoter:GUS transgenic seedlings were treated with NaCl, mannitol or ABA as mentioned in

d–f, using wild type as a negative control. Total RNA was isolated from these seedlings for quantitative RT-PCR analysis, using ACTIN2 as an internal control. Mean values and SE (bar) were shown from three independent experiments. Independent t tests demonstrated that there was significant (one asterisk: P < 0.05) or very significant (two asterisk: P < 0.01) difference in GUS or AtDi19-3 expression levels between the treated transgenic lines and the same lines grown on MS medium (controls). WT, wild type; AtDi19-3 promoter:GUS-L3 and -L5, AtDi19-3 promoter:GUS transgenic line 3 and 5. Scale bar 2 mm

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L6) was lower than that of wild type. About 55 % of wild type, and only 35 % of AtDi19-3 overexpression trans-genic seeds, but approximately 70 % of Atdi19-3 mutant seeds germinated after 3 days. Atdi19-3 mutant seeds

have successfully fully germinated, but the germinated AtDi19-3 overexpression transgenic seeds reached to only approximately 65 %, and wild type reached to about 85 % after 6 days (Fig. 4e).

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It is well known that ABA plays an essential role in salt-stress signaling (Leung and Giraudat 1998). We fur-ther tested whether AtDi19-3 was induced by exogenous ABA. On MS medium supplemented with 0.8 μM ABA, seed germination rate of Atdi19-3 mutant was significantly higher than that of wild type. On the contrary, germination of the AtDi19-3 overexpression seeds was more severely inhibited by ABA, compared with that of wild type. About 65 % of wild type seeds, and only 40 % of AtDi19-3 over-expression transgenic seeds, but approximately 80 % of Atdi19-3 mutant seeds germinated after 4 days (Fig. 4f).

On the other hand, the transgenic expression of AtDi19-3 in Atdi19-3 mutant under the control of AtDi19-3 promoter rescued the salt-, mannitol- and ABA-insensitive pheno-types of the mutant, similar to that of wild type (Fig. 4d–f), indicating that the phenotypes of Atdi19-3 mutant are indeed caused by the defect in AtDi19-3 gene. However, in the presence of 5 mM H2O2, seeds of all the AtDi19-3 lines germinated normally, like wild type (Fig. 4g).

AtDi19-3 is involved in response to salt, osmotic stress, ABA and H2O2 during early seedling development

To assess the effects of AtDi19-3 on seedling growth under salt, mannitol, ABA and H2O2 treatments, we used two approaches. One way is that seeds were directly planted in 200 mM NaCl-, 300 mM mannitol-, 1 μM ABA-, or 5 mM H2O2-containing MS medium to determine the sta-tus of seedling growth after germination (Fig. 5). Another is that seeds were germinated on MS medium for 48 h after stratification and then transferred to MS medium contain-ing 150 mM NaCl, 5 μM ABA, 300 mM mannitol or 5 mM H2O2 in the vertical position (Fig. 6). The results obtained with these two approaches were similar. There was no

significant difference among the different AtDi19-3 lines when seedlings grew on MS medium (Fig. 5b). However, seedlings of Atdi19-3 mutant grew better than those of wild type on NaCl-, ABA-, or mannitol-containing medium, but the AtDi19-3 overexpression seedlings were more sensi-tive to salt, ABA, and mannitol than wild type (Fig. 5c–e). The rate of cotyledon greening of Atdi19-3 mutant was sig-nificantly higher than that of wild type under NaCl, ABA, and mannitol treatments, whereas cotyledon greening of AtDi19-3 overexpression transgenic seedlings (L4 and L6) was remarkably inhibited by NaCl, ABA, and mannitol, compared with that of wild type (Fig. 5g–i).

When seedlings were transferred onto MS plates in the vertical position without any stress treatments, root growth of all AtDi19-3 lines was almost as same as that of wild type (Fig. 6a, f). However, when the seedlings were trans-ferred onto MS medium supplemented with 150 mM NaCl, 5 μM ABA or 300 mM mannitol for several days, growth of the primary roots of Atdi19-3 mutant was significantly superior to that of wild type, whereas root growth of the AtDi19-3 overexpression transgenic seedlings (L4 and L6) was inhibited much more than that of wild type (Fig. 6b–d). Under NaCl, ABA, and mannitol treatments, roots of Atdi19-3 mutant was much longer than those of wild type, but roots of AtDi19-3 overexpression seedlings was shorter than those of wild type (Fig. 6g–i). Furthermore, overex-pression of AtDi19-3 in the Atdi19-3 mutant could rescue its salt-, mannitol- and ABA-insensitive phenotype, similar to wild type (Figs. 5, 6).

Although H2O2 treatment had no effect on germination of all AtDi19-3 lines, Atdi19-3 mutant showed more sen-sitive to H2O2 during seedling development (Fig. 5f). The rate of cotyledon green of Atdi19-3 mutant grown on 5 mM H2O2-containing medium was only about half of wild type (Fig. 5j), and root length of Atdi19-3 mutant vertically cul-tured on MS medium with 5 mM H2O2 was nearly half of wild type (Fig. 6e, j).

Furthermore, the total chlorophyll content and proline content in leaves of both wild type and AtDi19-3 lines were determined under salt, mannitol and ABA treatments, respectively. Experimental results indicated that there was no significant difference in the total chlorophyll content and proline accumulation among AtDi19-3 lines grown in normal conditions (Fig. 7a, e). However, Atdi19-3 mutant had higher chlorophyll content and accumulated more pro-line, but the AtDi19-3 overexpression seedlings showed lower chlorophyll content and accumulated less proline than those of wild type after salt, mannitol and ABA treat-ments. Statistical analysis indicated that there were signifi-cant differences in chlorophyll content and proline content among Atdi19-3 mutant, AtDi19-3 overexpression trans-genic plants and wild-type under salt, osmotic and ABA

Fig. 4 Assay in seed germination of Arabidopsis AtDi19-3 lines under NaCl, mannitol, abscisic acid (ABA) and H2O2 treatments. a T-DNA insertion site in Atdi19-3 mutant. The T-DNA was inserted in the fourth intron of AtDi19-3 genomic DNA. Exons and introns are indicated by blank boxes and solid lines, respectively. b Quantita-tive RT-PCR analysis of AtDi19-3 expression in the wild type (WT), Atdi19-3 mutant (Atdi19), Atdi19-3 mutant complementation lines (Atdi19-3 + proAtDi19-3:AtDi19-3), and AtDi19-3-overexpression transgenic lines (35S:AtDi19-3oe). 10-day-old seedlings were used for RNA extraction. c–g Statistical analysis of seed germination rate. Seeds of wild type and transgenic lines were germinated on MS medium (c), MS medium with 150 mM NaCl (d), MS medium with 300 mM mannitol (e), MS medium with 0.8 μM ABA (f) and MS medium with 5 mM H2O2 (g), respectively. Mean values and SE (bar) were shown from three independent experiments (n > 60 seedlings per each line). WT wild type; Atdi19-3, Atdi19-3 mutant; Atdi19-3 + proAtDi19-3:AtDi19-3-L1 and -L5, Atdi19-3 mutant complemen-tation line 1 and 5; 35S:AtDi19-3oe-L4 and -L6, AtDi19-3-overex-pressing transgenic line 4 and 6. The assays were repeated three times along with three independent repetitions of the biological experiments

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stresses (Fig. 7b–d, f–h). These data imply that AtDi19-3 may be involved in plant response to salt, osmotic stress, ABA and H2O2 during early seedling development.

AtDi19-3 responds to drought stress

To investigate whether AtDi19-3 is involved in plant response to drought stress, water was withheld from 4-week-old AtDi19-3 lines and wild type plants for 10 days (Fig. 8). In this experiment, about 60 % wild type plants survived but almost wilted after 10 days under drought treatment, while all Atdi19-3 mutant plants remained rela-tively healthy survived, and nearly all the AtDi19-3 overex-pression transgenic plants (L4 and L6) died. The AtDi19-3 complementation lines (L1 and L5, under the control of AtDi19-3 promoter) showed a similar phenotype to wild type under drought-stress condition (Fig. 8a). Atdi19-3 mutant showed much greater drought-tolerance, whereas AtDi19-3 overexpression transgenic plants exhibited more drought-sensitivity than wild type. Subsequently, a leaf excision test was also performed among AtDi19-3 lines and wild type in order to further quantify drought toler-ance. Leaves were removed from each plant, weighed immediately, and weighed again after 1, 2, 3, 4 and 5 h, respectively. Any weight lost was considered to be due to water lost from the leaves. In the leaf excision test, water loss from Atdi19-3 mutant was significantly lower than that from wild type, while leaves of AtDi19-3 overexpres-sion transgenic plants lost more water than wild type. Furthermore, water loss from AtDi19-3 complementation lines was as same as wild type under dehydration condi-tions (Fig. 8b). These results suggest that AtDi19-3 may be involved in plant response to drought stress.

AtDi19-3 regulates stress-related and ABA-responsive genes

To investigate the mechanism of AtDi19-3 gene involved in abiotic stress and ABA signaling pathway, we analyzed expression levels of ABA-responsive genes (ABF4, ABI1, RAB18, and ERD15) and SOS2 (involved in SOS signaling pathway) served as markers for monitoring ABA and stress response pathways in Arabidopsis. As shown in Fig. 9, all the genes were strongly induced in AtDi19-3 lines under NaCl, mannitol and ABA treatments. Under normal condi-tions, the expressions of ABI1 and SOS2 were increased in the Atdi19-3 mutant, but reduced in the AtDi19-3 overex-pression transgenic lines, while the expression of RAB18 in the Atdi19-3 mutant and AtDi19-3 overexpression trans-genic lines was contrary to ABI1 and SOS2. Under exog-enous ABA treatment, transcript levels of ABF4, ABI1, RAB18, and ERD15 in wild type were remarkably lower than those in the AtDi19-3 overexpression transgenic lines, but higher than those in Atdi19-3 mutant. Salt stress induced down-regulation of ABI1, RAB18 and ERD15 in Atdi19-3 mutant, but up-regulation of those genes in AtDi19-3 overexpression transgenic lines. Under mannitol treatment condition, ABF4 and ABI1 transcripts accumu-lated more in Atdi19-3 mutant, whereas expression levels of RAB18 and ERD15 in Atdi19-3 mutant were lower than those in wild type (Fig. 9a–d). The abundance of SOS2 transcripts was dramatically increased in Atdi19-3 mutant but decreased in AtDi19-3 overexpression transgenic lines under normal, salt-, and mannitol-stress condition, and in the presence of exogenous ABA (Fig. 9e). These results suggest that AtDi19-3 protein as a transcriptional regulator may be involved in plant response to abiotic stress, as well as ABA and SOS signaling in plants.

Discussion

Cys2/His2-type (C2H2) zinc-finger proteins (such as RHL41, ST2, SCOF-1, OsISAP1, THZF1, DST, etc.) have been implicated in regulating plant defense response to biotic and abiotic stresses (Sakamoto et al. 2000, 2004; Lida et al. 2000; Kim et al. 2001; Mukhopadhyay et al. 2004; Xu et al. 2007; Huang et al. 2009). Although some gene families of above Cys2/His2-type zinc-finger pro-teins have been extensively analyzed in the last decade, the precise function of Di19 family members in plants still remains unknown as yet. Previous studies revealed that most of seven Arabidopsis AtDi19 s are induced early by dehydration and high salinity, and are phosphorylated in vitro by AtCDPK3 and AtCDPK11 (Sakamoto et al. 2000;

Fig. 5 Assay in cotyledon opening of AtDi19-3 transgenic Arabi-dopsis seedlings under NaCl, mannitol, abscisic acid (ABA) and H2O2 treatments. a The scheme shows the arrangements of wild type and transgenic lines in plates. b–f Growth status of wild type and transgenic seedlings grown on MS medium (b), MS medium with 200 mM NaCl (c), MS medium with 300 mM mannitol (d), MS medium with 1 μM ABA (e) and MS medium with 5 mM H2O2 (f) for 10 days, respectively. g–j Statistical analysis of cotyledon expan-sion and greening of seedlings grown on MS medium with 200 mM NaCl (g), MS medium with 300 mM mannitol (h), MS medium with 1 μM ABA (i) and MS medium with 5 mM H2O2 (j) for 10 days, respectively. Mean values and SE (bar) were shown from three inde-pendent experiments (n > 60 seedlings per each line). Independent t tests for equality of means demonstrated that there was (very) signifi-cant difference between wild type and transgenic plants (one asterisk: P value <0.05; two asterisk: P value <0.01). 1, wild type; 2, Atdi19-3 mutant; 3 and 4, Atdi19-3 mutant complementation line 1 and 5; 5 and 6, AtDi19-3-overexpressing transgenic line 4 and 6. The assays were repeated three times along with three independent repetitions of the biological experiments

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Fig. 6 Phenotypic analysis of Arabidopsis AtDi19-3 lines under NaCl, mannitol, abscisic acid (ABA) and H2O2 treatments. a–e 12-day-old seedlings of wild type, Atdi19-3 mutant, Atdi19-3 mutant complementation line, and AtDi19-3-overexpressing line growing on MS medium (a), MS with 150 mM NaCl (b), MS with 300 mM man-nitol (c), MS with 5 μM ABA (d) and MS with 5 mM H2O2 (e). f–j Statistical analysis of the relative root length of 12-day-old seedlings of wild type, Atdi19-3 mutant, Atdi19-3 mutant complementation line, and AtDi19-3-overexpressing line growing on MS medium (f), MS with 150 mM NaCl (g), MS with 300 mM mannitol (h), MS with

5 μM ABA (i) and MS with 5 mM H2O2 (j). After germination for 48 h, seedlings were transferred and grew for 12 days on MS medium without or with 150 mM NaCl, 300 mM mannitol, 5 μM ABA and 5 mM H2O2. Error bars represent SE of three replicates. Asterisk rep-resents (very) significant difference (one asterisk: P value <0.05; two asterisk: P value <0.01) between the transgenic lines and wild-type by t test. 1, wild type; 2, Atdi19-3 mutant; 3 and 4, Atdi19-3 mutant complementation line 1 and 5; 5 and 6, AtDi19-3-overexpressing transgenic line 4 and 6. The assays were repeated three times along with three independent repetitions of the biological experiments

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Milla et al. 2006). The seven AtDi19s genes were nearly expressed in all organs, although some differences were observed. AtDi19-3 transcript abundance was highest in rosette leaves and lowest in stems, but the opposite was observed for AtDi19-4 (Milla et al. 2006). Due to much low similarity of AtDi19-related proteins outside the zinc

finger domain and the different abilities/sites to binding to promoter/cis-acting element sites of the target genes, AtDi19s proteins may play diverse roles in plant devel-opment and in response to abiotic stress. A recent study revealed that Arabidopsis Di19-1 as a transcription fac-tor participates in response to drought stress by binding

Fig. 7 Assay of chlorophyll content and proline content in leaves of AtDi19-3 transgenic Arabidopsis seedlings. a–d Chlorophyll content in leaves of 10-day-old wild type and transgenic seedlings. Statistical analysis of chlorophyll content in leaves of seedlings grown on MS medium (a), MS with 150 mM NaCl (b), MS with 300 mM manni-tol (c) and MS with 5 μM ABA (d) for 10 days. e–h Proline content in leaves of 10-day-old wild type and transgenic seedlings. Measure-ment of proline content in seedlings of wild type and transgenic lines treated with 150 mM NaCl (f), 300 mM mannitol (g) and 5 μM ABA

(h) for 24 h, using MS medium (e) as control. Mean values and SE (bar) were shown from three independent experiments (n > 50 seed-lings per line). Independent t tests for equality of means demonstrated that there was very significant difference between wild type and transgenic plants (one asterisk: P value <0.05; two asterisk: P value <0.01). 1, wild type; 2, Atdi19-3 mutant; 3 and 4, Atdi19-3 mutant complementation line 1 and 5; 5 and 6, AtDi19-3-overexpressing transgenic line 4 and 6. The assays were repeated three times along with three independent repetitions of the biological experiments

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to the TACA(A/G)T element within the promoters of PR1 (pathogenesis-related 1), PR2, and PR5 genes, and di19-1 mutant showed a hypersensitive phenotype to drought stress compared with wild type (Liu et al. 2013). In con-trast, we observed Atdi19-3 mutant had greater drought-tolerance than wild type, and AtDi19-3 overexpression transgenic plants exhibited more sensitive to drought stress than wild type. Therefore, AtDi19-1 and AtDi19-3 may

respond to drought in different ways. Furthermore, cotton Di19 proteins (GhDi19-1 and GhDi19-2) are involved in plant response to abiotic stress (Li et al. 2010). For fur-ther investigating the function of the Di19s in plants under abiotic stress, we employed Atdi19-3 mutant (T-DNA insertion mutant of At3g05700, AtDi19-3) and AtDi19-3 overexpression transgenic Arabidopsis plants. Germi-nation and post-germination growth assays showed that

Fig. 8 Phenotypic analysis of Arabidopsis AtDi19-3 lines under drought stress. a Pheno-typic analysis of Arabidopsis plants under drought stress. Four-week-old plants (approxi-mately 30 plants of each line) were grown for 10 days with (Control, upper panel) or without (Drought, lower panel) irrigation. The experiments were repeated three times, with similar results. The scheme shows the arrangements of wild type and transgenic lines in pots. b Water loss measure-ments of leaves. Water loss from leaves excised from four-week-old plants at a specified time. Mean values and SE (bar) were shown from three independ-ent experiments (n = 20 fully expanded leaves per each line). WT wild type; Atdi19-3, Atdi19-3 mutant; Atdi19-3 + proAtDi19-

3:AtDi19-3-L1 and -L5, Atdi19-3 mutant complementation line 1 and 5; 35S:AtDi19-3oe-L4 and -L6, AtDi19-3-overex-pressing transgenic line 4 and 6. The assays were repeated three times along with three independent repetitions of the biological experiments

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Atdi19-3 mutant enhanced its tolerance to NaCl, manni-tol, and exogenous ABA, whereas AtDi19-3 overexpres-sion transgenic lines were more sensitive to salt, manni-tol and ABA than those of wild type. It is demonstrated that ABA maintains seed dormancy, prevents germination and inhibits seedling growth (Finkelstein et al. 2002), and that salt stress is able to induce ABA biosynthesis and trigger ABA-dependent signaling pathway (Zhu 2002). Therefore, we infer AtDi19-3 responds to salt stress in an ABA-dependent manner, although the transcript levels of AtDi19-3 were not induced by ABA. Overall, we suppose that AtDi19-3 acts a positive regulator in ABA response, whereas its role may be as a negative regulator in response to salt and drought stresses.

Reactive oxygen species (ROS) are known to play a role in the release of dormancy, although the mechanism through

which this operates is unknown (Bailly et al. 2008). Here, in the presence of exogenous H2O2, seeds of all the AtDi19-3 lines germinated normally as wild type, but Atdi19-3 mutant showed more sensitive to H2O2 during seedling develop-ment. This observation showed that AtDi19-3 may be involved in a ROS-mediated process, but whether it was also associated with ABA signaling can not yet be established.

Protein sequence analysis revealed that AtDi19-3 con-tains a conserved nuclear localization signal region (NLS) next to the two zinc finger domains in its sequence (Milla et al. 2006). Our data confirmed the nuclear localization of AtDi19-3 by GFP fluorescence assay. An autonomous gene activation test indicated that AtDi19-3 has transcrip-tion activation ability, and could bind to the TACA(A/G)T element, suggesting that AtDi19-1 may function as a tran-scription factor in plants, like AtDi19-1 (Liu et al. 2013).

Fig. 9 Quantitative RT-PCR analysis of expression of stress-related and ABA-responsive genes in Arabidopsis AtDi19-3 lines. 10-day-old seedlings of wild type, Atdi19-3 mutant, AtDi19-3 overexpres-sion transgenic lines and complementation lines were treated with or without 100 μM ABA, 150 mM NaCl and 300 mM mannitol for 6 h, respectively, and then total RNA was isolated from these seed-lings. Transcript levels of genes involved ABA signaling (ERD15, RAB18, ABI1 and ABF4) and SOS signaling (SOS2) were determined by quantitative RT-PCR, using ACTIN2 as a quantification control.

Error bars represent standard errors of three replicates. Independent t tests for equality of means demonstrated that there was very signifi-cant difference between wild type and transgenic plants (one asterisk: P value <0.05; two asterisk: P value <0.01). WT wild type; Atdi19-3, Atdi19-3 mutant; Atdi19-3 + proAtDi19-3:AtDi19-3-L1 and -L5, Atdi19-3 mutant complementation line 1 and 5; 35S:AtDi19-3oe-L4 and -L6, AtDi19-3-overexpressing transgenic line 4 and 6. The assays were repeated three times along with three independent repetitions of the biological experiments

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Motifs analysis of AtDi19-3 promoter with the PlantCARE analysis database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) revealed it contains ABRE and MBS cis-acting element, which involved in response to drought stress and ABA signaling. These elements are also present in the promoter regions of several dehydration responsive genes (such as AtMYB2 and AtMYC2) encoding the known transcriptional activators in ABA signaling (Abe et al. 2003). Moreover, histochemical assay of GUS expression revealed that activity of AtDi19-3 promoter was dramati-cally enhanced in cotyledons and true leaves, especially in vascular tissues after NaCl, mannitol and ABA treatments. Furthermore, quantitative RT-PCR analysis indicated that expression of AtDi19-3 gene in the transgenic plants and wild type with NaCl, mannitol and ABA treatments was remarkably stronger than those without NaCl, mannitol or ABA treatment. These results suggested that AtDi19-3 is involved in plant response to abiotic stress.

To investigate whether AtDi19-3 affects the expression of genes related to stress and ABA signaling, some marker genes were analyzed in AtDi19-3 lines. The results indi-cated that there were remarkable changes in expression levels of ERD15, RAB18, ABI1, ABF4 and SOS2 genes in the transgenic plants, compared with those in wild type, after NaCl, mannitol and ABA treatments, respectively. Previous study demonstrated that constitutive expression of ABF4 in Arabidopsis resulted in plant ABA-hyper-sensitivity and enhanced drought tolerance (Kang et al. 2002). ABA levels in the ABA-insensitive mutants, abi1-1 and abi2-1, are regulated by a feedback mechanism (Ver-slues and Bray 2006). In this study, ERD15, RAB18, ABI1 and ABF4 genes were up-regulated in the AtDi19-3 over-expression transgenic lines after ABA treatment, suggest-ing that AtDi19-3 as a positive regulator regulates those genes related to ABA signaling in Arabidopsis. In some cases, expression levels of stress-responsive genes cor-relate with stress tolerance in plants (Liu et al. 1998). In addition, SOS2 is an important signal transducer for plant salinity tolerance (Xiong et al. 2002). Here, the transcrip-tion levels of SOS2 was dramatically increased in Atdi19-3 mutant but decreased in AtDi19-3 overexpression transgenic lines under normal conditions, high salinity, osmotic stress and exogenous ABA. This may also help to explain the observation that Atdi19-3 mutant has stronger salt- and drought-tolerance than wild type. In addition, we have analyzed the promoter sequences of AtABF4, AtABI1, AtRAB18, AtSOS2 and AtERD15, and found that AtSOS2 and AtERD15 have one DiBS motif within their promoters, respectively. Therefore, it suggests Di19 may bind to the DiBS motifs in the promoters of AtSOS2 and AtERD15 in response to salt and drought stress. Based on the data presented in this study, we hypothesize that AtDi19-3 is a crucial transcriptional regulator in signaling

pathway for plant response to abiotic stress. AtDi19-3 may act a positive regulator to activate a subset of ABA-related genes in response to ABA signaling, but its role may be as a negative regulator in response to salt/drought stress.

Acknowledgments Authors thank Professor Jeff Harper in Univer-sity of Nevada (Reno, NV89557, USA) for kindly providing Atdi19-3 T-DNA insertion mutant seeds. This work was supported by the project from the Ministry of Agriculture of China for Transgenic Research (Grant No. 2014ZX08009-27B) and Natural Sciences Foun-dation of Hubei Province (Grant No. 2012FFA126, 2013CFA119).

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