Mol Biotechnol DOI 10.1007/s12033-014-9789-2

RESEARCH

A Novel Zinc-Finger HIT Protein with an Additional PAPA-1-like Region from Suaeda liaotungensis K. Enhanced Transgenic Arabidopsis Drought and Salt Stresses Tolerance Xiao-lan Li • Yu-xin Hu • Xing Yang Xiao-dong Yu • Qiu-li Li

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Ó Springer Science+Business Media New York 2014

Abstract Zinc-finger HIT belongs to the cross-brace zinc finger protein family and is involved in the regulation of plant defense and stress responses. In this study, we cloned a full-length zinc-finger HIT gene (1,377 bp) named SlPAPA1 using polymerase chain reaction from Suaeda liaotungensis K. and investigated its function by overexpression in transgenic Arabidopsis. SlPAPA1 contains a zinc-finger HIT domain and a Pim-1-associated protein-1 (PAP-1)-associated protein-1-like (PAPA-1-like) conserved region. Its expression in S. liaotungensis was induced by drought, high-salt, and cold (4 °C) stresses and by abscisic acid (ABA). Subcellular localization experiments in onion epidermal cells indicated that SlPAPA1 is localized in the nucleus. Yeast-one hybrid assays showed that SlPAPA1 functions as a transcriptional activator. SlPAPA1 transgenic Arabidopsis displayed a higher survival ratio and lower rate of water loss under drought

Electronic supplementary material The online version of this article (doi:10.1007/s12033-014-9789-2) contains supplementary material, which is available to authorized users. X. Li  Y. Hu  X. Yang  X. Yu  Q. Li (&) College of Life Sciences, Liaoning Normal University and Key Laboratory of Plant Biotechnology of Liaoning Province, 1 South Liushu Street, Ganjingzi District, Dalian 116081, Liaoning, China e-mail: [email protected] X. Li e-mail: [email protected] Y. Hu e-mail: [email protected] X. Yang e-mail: [email protected] X. Yu e-mail: [email protected]

stress; a higher germination ratio, higher survival ratio, and lower root inhibition rate under salt stress; and a lower germination ratio and root inhibition rate under ABA treatment, compared with wild-type Arabidopsis. These results suggested that SlPAPA1 functions as a stressresponsive zinc-finger HIT protein involved in the ABAdependent signaling pathway and may have potential applications in transgenic breeding to enhance crops abiotic stress tolerances. Keywords Abiotic stress  Zinc finger HIT  PAPA-1like  Transgenic plant  Suaeda liaotungensis K Abbreviations ABA Abscisic acid bZIP Basic leucine zipper CBF Core binding factor DEAD The amino acid sequence Asp-Glu-Ala-Asp DREB Dehydration response element binding factor FON ZNHIT2 protein (zinc finger HIT domaincontaining protein 2), encoded by the C11orf5 gene on chromosome 11q13–q22 from Homo sapiens GFP Green fluorescent protein HIT High temperature IGFBPs IGF-binding proteins MODY Maturity-onset diabetes of the young MYB Myeloblastosis NAC NAM (No Apical Meristem), ATAF1 or ATAF2 and CUC2 (cup-shaped cotyledon) ORF Open reading frame PAPA-1 Pim-1-associated protein-1 (PAP-1)associated protein-1 PEG Polyethylene glycol 6000 6000

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PHD RSGI qRT-PCR zf-HIT

Plant homeodomain RIKEN structural genomics/proteomics initiative Quantitative real-time polymerase chain reaction Zinc-finger HIT

Introduction Zinc finger proteins form a large family of proteins involved in numerous activities of plant growth and development and are known to regulate resistance mechanism for various biotic and abiotic stresses [1, 2]. The majority of zinc finger proteins bind to DNA (and also to RNA in the case of TFIIIA), thereby playing important roles in transcriptional and translational processes [3]. The zinc finger proteins are classified into nine types: C2H2, C8, C6, C3HC4, C2HC5, C4, C4HC3, and CCCH (C and H represent cysteine and histidine, respectively) [2]. Many zinc finger proteins from plant have been reported in abiotic stress resistance. These proteins include AtSF1 (CCCH), AtSF2 (CCCH), AtC3H49/AtTZF3 (CCCH), and AtC3H20/AtTZF2 (CCCH) of Arabidopsis [4, 5], GhZFP1 (CCCH), GhDi19-1 (C2H2), and GhDi19-2 (C2H2) of cotton [6, 7], ZFP182 (C2H2), ZFP179 (C2H2), RZF71 (C2H2) of rice [8–10], StZFP1 (C2H2) of potato [11], PSTZ (C2H2) of Populus euphratica [12], SCTF-1 (C2H2) of soybean [13]. These reports indicate that zinc finger proteins have important role in plant stress resistance. The zinc finger HIT (Zf-HIT) domain is a sequence motif found in many proteins, which contains up to six cysteine residues that could coordinate zinc. Zf-HIT was named after the first protein that originally defined the domain: the yeast HIT1 protein [14]. The function of this domain is unknown, but it is mainly found in nuclear proteins involved in gene regulation and chromatin remodeling [15, 16]. Zf-HIT belongs to the cross-brace zinc finger protein family, including the B-box, RING, and PHD domains, with the same interleaved zinc-binding mode. A search of the Pfam database [17] revealed that the Zf-HIT containing proteins (total 121) are mainly divided into three groups: 76 % of the Zf-HIT proteins contain a single Zf-HIT domain without any other annotated domains; 12.4 % of the Zf-HIT proteins have an additional PAP-1-associated protein-1 (PAPA-1) homology sequence; and 11.6 % have an additional DEADbox helicase domain [16]. SlPAPA1, studied in this paper, belongs to the second group of Zf-HIT containing proteins. Environment stresses, such as drought, high-salinity, and cold, reduce productivity and cause significant crop losses globally. Drought and salinity affect more than 10 %

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of arable land, which results in more than 50 % decline in the average yields of important crops worldwide [18]. Tolerance or susceptibility to these stresses is a complex process, as stress may affect multiple stages of plant development and often several stresses concurrently affect the plants [19]. Therefore, plants have evolved a series of mechanisms to survive such environmental adversities. Plant stress tolerance involves changes at whole-plant, tissue, cellular, physiological, and molecular levels. Molecular responses to abiotic stress include perception, signal transduction, gene expression and, ultimately, metabolic changes in the plant, resulting in stress tolerance [20]. Transcription factors (TFs) and their corresponding cis-regulatory sequences act as molecular switches for gene expression, regulating their temporal and spatial expression. Increasing evidence demonstrates that numerous TFs, such as DREB, CBF, bZIP, zinc-finger, MYB, and NAC, are directly or indirectly involved in the regulation of plant defense and stress responses [21–27]. However, there is no information on the abiotic stressrelated Zf-HIT proteins in plants. Suaeda liaotungensis K, a halophytic plant, as a source of genetic material for introduction into non-salt-tolerant crops, grows widely by the seashore in Dalian, China. We discovered a sequence annotated as a predicted protein with a Zf-HIT domain and a PAPA-1-like conserved region from the database of S. liaotungensis transcriptome sequencing, and named this protein SlPAPA1. To identify whether SlPAPA1 was related to abiotic stress, we designed preliminary tests and found that SlPAPA1 was induced by salt stress. In this study, we cloned SlPAPA1 and characterized its function. The expression of SlPAPA1 was induced by drought, high salt, cold, and abscisic acid (ABA), as assessed by realtime quantitative polymerase chain reaction. Its overexpression improved the drought and high-salt stress tolerance of transgenic plants. Furthermore, SlPAPA1 is localized in the nucleus and functions as a transcriptional activator, as determined by subcellular localization and transactivation assays. Therefore, SlPAPA1 may provide a new clue to abiotic stress resistance research and is expected to be used in genetically modified crops.

Materials and Methods Plant Materials Green leaves and seeds of wild S. liaotungensis were collected from the seashore in Dalian. The leaves were used for SlPAPA1 cDNA cloning. The seeds were used to breed seedlings for gene expression analysis. Arabidopsis thaliana accession Columbia (Col-0) was used for gene transformation and as a phenotypic control,

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and was grown in a controlled environment chamber at 22 °C, with a 16/8 photoperiod, light intensity of 120–150 lmol/m2/s, and 60 % relative humidity. SlPAPA1 Isolation and Sequence Analysis The sequence of SlPAPA1 was obtained from the database of S. liaotungensis transcriptome sequencing. It was 1,914-bp long and contained a full-length open reading frame (ORF). To obtain the ORF by the reverse transcription polymerase chain reaction (RT-PCR) approach, two primers were used (sense: 50 -ATGGATTGTCTCAATGATCTTGCA-30 and antisense: 50 -CAG CAAGCAGGCAGAAGGT-30 ). The PCR conditions for amplifying SlPAPA1 were as follows: 3 min pre-denaturation at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min at 72 °C; and a final extension for 10 min at 72 °C. The PCR product was purified and cloned into the pEASY-T1 cloning vector (TRANSGEN BIOTECH, China) for sequencing (TAKARA, Japan). Alignment of SlPAPA1 and other proteins with PAPA-1like conserved regions and Zf-HIT domains was performed using the ClustalX program (version 1.83) and viewed by GeneDoc software (version 2.5). A phylogenetic tree was constructed using the MEGA program (version 4.0) by the neighbor-joining method. The parameters of pairwise deletion and the p-distance model were used. A bootstrap test of phylogeny was performed with 1,000 replicates. Expression Analysis of SlPAPA1 under Different Treatments Seedlings (4 weeks old) of S. liaotungensis were used for the various stress treatments. The treatments were executed at 25 °C and 50–65 % humidity and the seedlings were grown in 1/10 MS culture medium with 200 mM NaCl, 100 lM ABA, and 10 % PEG 6000 (polyethylene glycol 6000), respectively. For cold treatment, seedlings were transferred to a growth chamber at a temperature of 4 °C. For gene expression analysis, total RNA was isolated from liquid nitrogen-frozen seedlings using RNAiso Plus reagent (TAKARA, Japan), according to the protocol of manufacturer. RNA integrity was verified by 1 % agar gel electrophoresis. The first-strand cDNA was synthesized with 1 lg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (TAKARA, Japan), according to the manufacturer’s procedure. Primers were designed using Beacon Designer version 7.0 (Premier Biosoft international, Palo Alto, CA, USA) with melting temperatures of 60–65 °C, primer lengths of 20–24 bp, and amplicon lengths of 90–200 bp. The primers of SlPAPA1 and the internal reference gene SlActin (a S. liaotungensis actin gene, GenBank accession number: JX860282.1) used for qRT-PCR were 50 CAGCAAATCGTATGTGTGTGGA-30 and 50 -ATCTG

TTCCTTGTGCCCTTGAGTT-30 , and 50 -ATCCCAAGG CTAATCGTGAAAA-30 and 50 -CACCATCAC CAGAGT CCAACA-30 , respectively. qRT-PCR was conducted on a Thermal Cycler Dice Real Time System TP800 (TAKARA, Japan) using SYBR Premix Ex Taq II (Tli RnaseH Plus) (TAKARA, Japan). Baseline and threshold cycles (Ct) were automatically determined using the Thermal Cycler Dice Real Time System TP800 Software release Version 3.0. Relative gene expression with respect to the internal reference gene SlActin1 (an actin gene, GenBank accession number: JX860282.1) was determined as described previously, using the 2 (-DDCT) method [28]. Subcellular Localization of SlPAPA1 The full-length ORF of SlPAPA1 was amplified with the primers 50 -gcGAATTCATGGATTGTCTCAATGATCTT GCA-30 and 50 -gcGGATCCTCAGCAAGCAGGCAGAAG G-30 , digested with EcoRI and BamHI, and fused to the 30 end of GFP in the pEGAD vector to generate the fusion construct 35S::GFP-SlPAPA1. The fusion gene and the negative control pEGAD vector were transformed into living onion epidermal cells by biolistic bombardment with a GeneGun (GJ-1000; China), according to the instruction manual (helium pressure, 9 MPa). Protein expression was observed under a confocal laser-scanning microscope (Ti-E; Nikon). Transactivation Assay of SlPAPA1 The entire coding region of SlPAPA1 was fused to the GAL4 DNA-binding domain in the pGBKT7 vector. According to the manufacturer’s protocol (Stratagene, USA), pGBKT7-SlPAPA1 and the negative control pGBKT7 vector were transferred into the yeast strain AH109, respectively. The transformed yeast cells were grown on SD glucose medium lacking Trp, and medium lacking Trp, His, and Ade, respectively. The transactivation activity of each protein was evaluated according to its growth status and b-galactosidase filter lift assay (Yeast Protocols Handbook; Clontech, USA). Abiotic Stress Assays of Transgenic Arabidopsis Plants The transformation vectors harboring the 35S::GFP or 35S::GFP-SlPAPA1constructs were introduced into Agrobacterium tumefaciens (GV3101) and then transferred into wild-type (Wt) Arabidopsis by the floral dip transformation method [29]. Positive transgenic lines of T1 and T2 generations were screened on MS agar medium containing 37.5 lM Basta. The T2 generation was selected according to its segregation ratio (resistant: sensitive = 3:1), and confirmed by genomic PCR. The selected transgenic lines (pEGAD-

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SlPAPA1-1 and pEGAD-SlPAPA1-2, OE1 and OE2) that displayed the segregation ratio were chosen for further analysis. Expression levels of SlPAPA1 were determined by semi-quantitative RT-PCR. Total RNA samples were extracted from appropriate plant materials using the RNAiso Plus kit (TAKARA, Japan). Synthesis of first cDNA, RTPCR was performed as described above. Expression levels were quantitated by agars gel electrophoresis. T2 generation seeds were sterilized, suspended in 0.1 % agar and plated on MS medium (including 37.5 lM Basta), then stratified in darkness at 4 °C for 2 days and transferred to growth chambers with the same environmental conditions described above. Ten-day-old seedlings were transferred from MS plates to water-saturated soil for 7 days, water was withheld from one group until the plants showed evident drought-stressed phenotypes and then the plants were rewatered for drought stress treatment. A second group was watered with 200 mM NaCl solution until the plants showed evident salt-stressed phenotypes for high-salt stress treatment. A third group was grown for another 2 weeks, was treated at -20 °C for 90 min, and transferred to normal conditions. For the leaf water loss assay, rosette leaves excised from plants grown in soil were placed on filter paper for air-drying treatment. The leaf weight was measured over a series of time points. To conduct the root growth assay, seeds sown on MS plates were stratified for 2 days at 4 °C and grown vertically for 3 days under normal conditions, before the seedlings were transferred to vertical square MS plates with ABA (10 lM) or NaCl (200 mM). To conduct the germination rate assay, seeds of transgenic lines (OE1 and OE2) and control lines (wild-type and vector control) were placed on MS medium without or with ABA (10 lM), NaCl (200 mM) and Basta (37.5 lM), respectively. Seeds were vernalized at 4 °C in the dark for 3 days, and then transferred to growth chambers. The germination ratio was calculated on the sixth day.

its C terminus (Fig. 1a) (GenBank accession number KC176075). Phylogenetic analysis revealed that SlPAPA1 was clustered into the Eudicots subgroup (see Appendix S1 in Supporting Information). Multiple alignments with PAPA-1 proteins from other species confirmed the presence of the PAPA-1-like region and the Zf-HIT domain which contains four cysteine residues in SlPAPA1 (Fig. 1b). The proteins used in the alignment and phylogenetic tree all had these two domains and were obtained by database searching in NCBI. SlPAPA1 is a Potential Stress-Related Gene To identify whether SlPAPA1 could be induced by abiotic stresses, the expression pattern of SlPAPA1 in different tissues of wild S. liaotungensis and under various abiotic stress treatments in young leaves was investigated using qRT-PCR. The results showed that the expression of SlPAPA1 was the highest in the leaves (Fig. 2a), and that SlPAPA1 was induced by drought (10 % PEG 6000) (Fig. 2b), high salt (200 mM NaCl) (Fig. 2c), cold (4 °C) (Fig. 2d), and 100 lM ABA (Fig. 2e). The expression patterns and maximum expression levels differed for each stress treatment. The relative expression levels peaked at 3 h for drought, 6 h for NaCl, 24 h for cold, and 12 h for ABA; with the corresponding maxima being 6.13-, 5.40-, 3.69-, and 6.99-fold greater than the control (0 h), respectively. SlPAPA1 Functions as a Transcriptional Activator and is Localized in the Nucleus

Results

To determine the subcellular localization of SlPAPA1, a GFPSlPAPA1 fusion construct and the GFP control in pEGAD driven by the CaMV 35S promoter were transiently expressed in onion epidermal cells and analyzed by confocal laser scanning microscopy. Figure 3(a–c) shows that the GFP-SlPAPA1 fusion protein was targeted to the nuclei of the cells, while the control GFP protein was located in the cytoplasm (Fig. 3d–f). This result indicated that SlPAPA1 was a nuclear protein. The yeast one-hybrid system was used to identify a possible transcriptional activation function of SlPAPA1. As shown in Fig. 4, all transformants grew well on SD/Trpmedium. However, only transformants containing pGBKT7SlPAPA1 could grow on SD/Trp-/His-/Ade-medium and showed b-galactosidase activity that cells containing pGBKT7 could not (Fig. 4). These results indicated that SlPAPA1 functioned as a transcriptional activator.

SlPAPA1 Encodes a Protein with a PAPA-1-like Conserved Region and Zf-HIT Domain

Overexpression of SlPAPA1 Confers Drought Tolerance on Transgenic Arabidopsis

The SlPAPA1 gene encodes a 459-amino-acid protein with a conserved PAPA-1-like region and a Zf-HIT domain at

We showed that SlPAPA1 is a drought-inducible gene (Fig. 2b); therefore, it is likely to regulate drought

Statistical Analysis For qRT-PCR, the 2 -DD CT method was used to calculate relative expression levels of the target gene in stressed and non-stressed leaves and other tissues. Significant differences in relative expression levels were identified using a one-way analysis of variance test (P \ 0.05; n = 3) using SPSS 13.0 (SPSS Inc., USA).

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Fig. 1 Multiple alignment and structure of SlPAPA1. a Structure of SlPAPA1. A PAPA-1-like conserved region annotation with a PAPA1 gray box and a Zf-HIT domain annotation with a Zf-HIT box at the C-terminus. b Multiple alignment of PAPA-1-like conserved region and Zf-HIT domains from different species. Identical amino acid residues are highlighted in purple. The regions annotated indicate the conserved PAPA-1-like region and the Zf-HIT domain. Species

corresponding to the accession numbers were as follows: Vitis vinifera, CAN72174.1; Fragaria vesca subsp. Vesca, XP_004295088.1; Prunus persica, EMJ25181.1; Populus trichocarpa, XP_002311388.1; Ricinus communis, XP_002519215.1; Theobroma cacao, EOY06706.1; Cicer arietinum, XP_004508152.1; Arabidopsis thaliana, AAF63832.1; and Arabidopsis lyrata subsp. Lyrata, XP_002882487.1

signaling. To characterize such a function, a whole-plant drought assay was performed in soil using T2 generation transgenic lines to test SlPAPA1expression levels (Fig. 5a). Seedlings (10 days old) were transferred from MS plates to water-saturated soil for 7 days, and then water was withheld to cause a severe water deficit in soil. After 21 days, most of the Wt, pEGAD (vector control (V)), and pEGADSlPAPA1 (OE1, OE2) plants had wilted because of the extreme water deprivation (Fig. 5b). After re-watering, 75–78 % of pEGAD-SlPAPA1 plants continued to grow and successfully produced seeds, whereas most of the other plants (Wt and V) did not recover and died from drought, showing survival ratios of 25 % for Wt and 33 % for V (Fig. 5c). This experiment was repeated three times with similar results. The results showed that high-level expression of SlPAPA1 could enhance plant drought tolerance. Regulation of transpiration plays a vital role in plant response to drought stress. To address whether the SlPAPA1-related enhanced drought tolerance was associated with transpiration, we compared the wilting phenotype of detached leaves from different plants that were subjected to air-dry treatment at 25 °C with 55 % humidity. After 2 h, obvious phenotypic changes were observed in these leaves

(Fig. 5d). At this time point, only the leaves from OE1 and OE2 plants retained a normal shape and only showed a slightly wilting phenotype, whereas the leaves of other plants were severely wilted. Similar phenomena were observed after 4 h of treatment (Fig. 5d). We further addressed the reasons for these phenotypic alterations by measuring water loss ratios in detached leaves from these plants (Fig. 5e). In this assay, we found that the leaf weight of OE1 and OE2 plants decreased at a slow rate comparable to the water loss rates. This quantitative result was consistent with the phenotypic observation in the leafwilting assay. Overexpression of SlPAPA1 Enhanced High Salinity Tolerance of Transgenic Arabidopsis SlPAPA1 is induced by salt (Fig. 2c); therefore, we asked whether this induction correlated with the plant’s response to salinity. As shown in Fig. 6a, the germination rates (radical emergence) of OE1 and OE2 seeds were slightly higher than Wt and V plants on MS medium with 200 mM NaCl. The germination rates of all untreated plants were the same (data not shown). To further correlate SlPAPA1

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Fig. 2 The expression patterns of SlPAPA1. a The relative expression level of SlPAPA1 was measured in leaves, stems, and roots of S. liaotungensis using qRT-PCR. b The relative expression level of SlPAPA1 was measured in leaves under drought stress. c The relative expression level of SlPAPA1 was measured in leaves under salt stress. d The relative expression level of SlPAPA1 was measured in leaves under cold stress. e The relative expression level of SlPAPA1 was measured in leaves under ABA treatment using qRT-PCR. Sevenweek-old S. liaotungensis plants were treated with 10 % PEG 6000 (drought), 200 mM NaCl (salt), 100 lM ABA, and exposed to 4 °C

(cold) for 0.5, 1, 3, 6, 12, and 24 h. Standard errors were calculated from three biological replicates in which SlActin1 (an actin gene, accession number JX860282.1) transcripts were used as internal controls. The 2-44CT method was used to measure the relative expression levels of the target gene in stressed and non-stressed leaves. Error bars represent standard error. *, **Significantly different from expression level of control (0 h): *P \ 0.05; **P \ 0.01; lowercase indicates significant differences between various tissues based on a one-way analysis of variance test (P \ 0.05; n = 3)

Fig. 3 Subcellular localization assay of SlPAPA1 in onion epidermal cells. Onion epidermal cells were bombarded with constructs carrying 35S::GFP (Vector) or 35S::GFP-SlPAPA1 by a GJ-1000 Gene gun. 35S::GFP and 35S::GFP-SlPAPA1 fusion proteins were transiently

expressed in onion epidermal cells and observed using a Nikon Ti-E laser scanning confocal microscope. Images are dark fields for green fluorescence (a, d), bright field (b, e), and merged (c, f). Bar 50 lm

function with plant sensitivity to NaCl during the postgermination stage, root elongation was analyzed for these plants. Seedlings (4 days old) were transferred to MS

medium with or without 200 mM NaCl. After 7 days, growth retardation was observed for OE1 and OE2 on MS medium; however, the retardation was not dramatic

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Fig. 4 Transactivation activity analysis of SlPAPA1 in yeast. A fusion protein of the GAL4 DNA-binding domain and full-length SlPAPA1 was checked for its transactivation activity in yeast strain

AH109. The pGBKT7 vector was used as a negative control. Experiments were repeated three times independently, and the results were consistent

Fig. 5 SlPAPA1 expression in transgenic plants and droughttolerance assays of SlPAPA1 overexpression plants. a SlPAPA1 expression in transgenic plants. b Photographs taken when the drought-stress phenotypes appeared and 3 days after re-watering. c Percentage of surviving plants in droughttolerance assays; bars indicate standard errors. d Comparison of phenotypes of excised leaves under air-drying treatment for 0, 2, and 4 h. Bar = 1 cm. e Leaf water loss assay. Leaf weights were measured at the indicated time points (n = 9). Curves were drawn based on the data from three independent experiments. Wild-type (Wt) and pEGAD (V) lines were used as control plants.OE1 and OE2 represent pEGAD-SlPAPA1-1 and pEGAD-SlPAPA1-2 plants, respectively. Error bars represent standard error. *, **Significantly different from Wt: *P \ 0.05; **P \ 0.01

compared with Wt and V plants on MS medium with 200 mM NaCl (Fig. 6b, c). To characterize the function of SlPAPA1 in salt tolerance, a whole-plant salt tolerance assay was performed in soil. Seedlings (10 days old) were transferred from MS plates to water-saturated soil for 7 days, and were then

watered with 200 mM NaCl solution until severe saltstressed phenotypes appeared (Fig. 6d). Most of the Wt and V plants died, showing low survival ratios (44.44 % for Wt, 33.33 % for V); but 88 % of the OE1 and OE2 lines continued to grow (Fig. 6e). The experiment was repeated three times with similar results. These results indicated that

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Fig. 6 High-salinity assays of SlPAPA1 overexpression plants. a Germination rates analysis. Seeds of wild-type (Wt), pEGAD (V), pEGAD-SlPAPA1-1 (OE1), and pEGAD-SlPAPA1-2 (OE2) lines were placed on MS medium containing 200 mM NaCl and germinated for 3 days. Percentages are means (n = 40–60 each) of three repeats ± SE. b Phenotypic comparison of root lengths. Wt, V, and OE1 and OE2 seeds were germinated and grown on MS medium with

or without 200 mM NaCl for 7 days. c Statistical comparison of root lengths. d Photographs of the salt-stressed phenotypes. e Percentage of survival plants in salt-tolerance assays; bars indicate standard errors. Wt and V lines were used as control plants. Error bars represent standard error. *, **Significantly different from Wt: *P \ 0.05; **P \ 0.01

high levels of expression of SlPAPA1 could enhance plant salt tolerance, but results in root elongation inhibition of transgenic plants under normal conditions.

After 7 days, root lengths of OE1 and OE2 were shorter than Wt and V on MS medium without ABA, but the root length of the four lines was similar on MS medium with 10 lM ABA(Fig. 7b, c). Taken together, these results indicate that SlPAPA1 modulates plant ABA signaling differently in the germination stage and root elongation stage; high SlPAPA1 expression contributes to ABA hypersensitivity in transgenic Arabidopsis during germination, but the sensitivity was declining during the root elongation stage.

SlPAPA1 Overexpression Affects Transgenic Arabidopsis Sensitivity to ABA The expression of SlPAPA1 was dramatically induced by ABA, as assessed by RT-qPCR (Fig. 2e), suggesting the potential function of SlPAPA1 in the ABA response. To elucidate the role of SlPAPA1 in ABA signaling, OE1, OE2, Wt, and V plants were germinated on MS plates with 10 lM ABA for 3 days. The high concentration of ABA (10 lM) used in this study led to extreme growth inhibition for all the plants; however, OE1 and OE2 plants showed the most severe arrested growth under these conditions, as reflected by lower germination rates. By contrast, the germination rates of Wt and V plants were higher (Fig. 7a). To further correlate SlPAPA1 function with plant sensitivity to ABA during the post-germination stage, root elongation was analyzed for these plants. Seedlings (4 days old) were transferred to MS medium with or without 10 lM ABA.

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Discussion SlPAPA1 may be a Transcription Factor TFs are proteins that act together with other transcriptional regulators, including chromatin remodeling/modifying proteins, to encourage or obstruct the access of RNA polymerases to the DNA template [30]. Zf-HIT domains are mainly found in nuclear proteins involved in gene regulation and chromatin remodeling. The human thyroid

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Fig. 7 Sensitivity of SlPAPA1 overexpression lines to ABA. a Germination rates of SlPAPA1 overexpression lines on MS medium with 10 lM ABA. b, c Phenotypic and statistical comparisons of root lengths of wild-type, pEGAD (V), pEGAD-SlPAPA1-1 (OE1), and

pEGAD-SlPAPA1-2 (OE2) lines in the presence of MS with or without 10 lM ABA. Error bars represent standard error. *, **Significantly different from Wt: *P \ 0.05; **P \ 0.01

hormone receptor interacting protein 3 (TRIP-3, ZNHIT3) comprises 155 amino acids and has a characteristic N-terminal Zf-HIT domain spanning residues 1–52. TRIP-3 is a co-activator associated with hepatocyte nuclear factor-4a, which is a transcription factor and a member of the steroid hormone receptor superfamily [15]. Despite the medical importance of the Zf-HIT-containing proteins, their role in plants remains unknown. In this study, we found that the SlPAPA1 gene encodes a protein with a conserved PAPA-1-like region and a Zf-HIT domain at its C terminus (Fig. 1a). Sequence alignment also revealed the characteristic conserved amino acid sequences (Fig. 1b). Subcellular localization experiments in onion epidermal cells indicated that SlPAPA1 was localized in the nucleus (Fig. 3), and transactivation assays demonstrated that SlPAPA1 functioned as a transcriptional activator (Fig. 4). Although the Zf-HIT domain in protein FON is not a DNA-binding domain [16], SlPAPA1 had transcriptional activation activity, perhaps mediated through its PAPA-1-like region, which was also found in a nucleolar protein PAPA-1 [31]. These results may support the hypothesis that SlPAPA1 is a novel member of the zinc finger protein family and a transcription factor.

finger [32, 33]. CgZFP1, a C2H2 type zinc finger protein gene from Chrysanthemum, is an important regulator involved in the salt and drought stress response in plants [34]. TaCHP, a CHP-rich zinc finger protein from Triticum aestivum, is involved in the plant response to abiotic stress by the ABA-dependent and ABA-independent signaling pathways [33]. GhDi19-1 and GhDi19-2 of cotton are involved in the plant response to salt/drought stress and ABA signaling [7]. StZFP1, a typical TFIIIA-type twofinger zinc finger gene, might be involved in potato’s responses to salt and dehydration stresses through an ABAdependent pathway [11]. However, the function of Zf-HIT domain containing proteins under abiotic stress was unknown. Here, we found that the expression of SlPAPA1, which encodes a novel member of the zinc finger family proteins with a zinc finger domain and a PAPA-1-like conserved region, was induced by drought, high salt, cold, and ABA (Fig. 2b–e). To further identify the function of SlPAPA1 in response to abiotic stresses, we overexpressed SlPAPA1 in transgenic A. thaliana. The transgenic plants showed improved drought and high-salt tolerance, as assessed by a whole-plant abiotic stress assay (Figs. 5b, 6d). In addition, analysis of the transpiration rate of detached leaves indicated that the rate of water loss of the transgenic plants was lower than that of Wt and V plants (Fig. 5d, e). Meanwhile, the survival rate of transgenic plants was higher than that of Wt and V plants (Fig. 5c). These data provided further evidence that SlPAPA1 can enhance transgenic plants drought tolerance. Similarly, we analyzed the germination rate, root elongation, and survival rate of SlPAPA1 transgenic plants, Wt, and V plants under NaCl treatment. The germination ratio and survival rate assay showed that the seeds of the transgenic plants had slightly higher germination rates and a dramatically higher survival rate compared with Wt and V plants under high-salt stress (Fig. 6a, e). Moreover, root

SlPAPA1 Functions in ABA Signaling and Abiotic Stress Responses ABA is a broad-spectrum phytohormone involved not only in regulating growth, and development, but also in coordinating various stress signal transduction pathways in plants during abiotic stresses. The plant response to abiotic stresses involves both ABA-dependent and ABA-independent signaling pathways. The ABA-dependent and ABAindependent signal transduction pathways from stress signal perception to gene expression involve different TFs, such as DREB, MYC/MYB, AREB/ABF, NAC, and zinc

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elongation assays indicated that overexpression of SlPAPA1 led to root growth retardation in transgenic plants under normal conditions, but this retardation was not dramatic compared with Wt and V plants under high-salt treatment. On the contrary, the root lengths of transgenic plants were longer (Fig. 6b, c). This evidence suggested that SlPAPA1 transgenic plants had high-salt tolerance. To further elucidate the role of SlPAPA1 in ABA signaling, the germination ratio and root elongation were also tested under ABA treatment. The results showed that overexpression SlPAPA1 resulted in a significantly lower germination rate of transgenic plants seeds compared with Wt and V plants under ABA treatment, which indicated that high SlPAPA1 expression contributes to ABA hypersensitivity in Arabidopsis during germination. However, this sensitivity was noticeably high during the root elongation stage; the root retardation of transgenic plants was not as dramatic as that observed for Wt and V plants (Fig. 7). This suggested that SlPAPA1 might function in the ABA-dependent signaling pathway. We also tested whole-plant cold stress tolerance; however, the transgenic plants showed a similar phenotype to the Wt plants (see Appendix S3 in Supporting Information). Taken together, the results of the present study revealed that transcription of SlPAPA1 was induced by various abiotic stresses, such as high salt, cold, drought, and ABA, and overexpression of SlPAPA1 enhanced the tolerance of transgenic plants to high salt and drought. SlPAPA1 may function in the ABA-dependent signaling pathway. SlPAPA1 is an Important Gene and has Significant Research Value Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. They have an extraordinary diversity of structure and function. Based on previous research, their functions include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding [3]. The function of Zf-HIT domain is not clear, with only a few reports in the medical field [15]. In particular, functional research on proteins with this domain and a PAPA-1like conserved region in plants is lacking. However, phylogenetic analysis indicated that this type of protein is widespread in green plants (see Appendix S1 in Supporting Information), and multiple sequence alignment showed that these two domains are relatively conserved (Fig. 1b). The Zf-HIT domain in protein FON is not a DNA-binding domain [16]; however, our study showed that SlPAPA1 functioned as a transcriptional activator (Fig. 4). Furthermore, SlPAPA1 responds to abiotic stresses and its overexpression enhanced the drought and salt tolerance of transgenic plants. Further research is required to determine whether the PAPA-l-like domain in SlPAPA1 is

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responsible for its response to biotic stresses. This region is found in the PAPA-1 superfamily of proteins. PAPA-1 is a nuclear-binding partner of IGFBP-2 that modulates its growth-promoting actions. IGFBP-2 and PAPA-1 were colocalized predominantly in the nucleus [35]. Thus, functional research of this type of gene has important biological significance, and they may represent a new resource for stress tolerance research in plants. Further study of SlPAPA1 will provide important information on the functions of these genes. Acknowledgments We would like to thank Dr Shaoming Tong (Liaoning Normal University) for kindly providing the pEGAD vector and seeds of Arabidopsis thaliana and Dr Qiao Su (Dalian University of Technology) for providing the GeneGun (GJ-1000, China). This work was supported by grants from the National Natural Science Foundation of China (No. 30871389) to Professor Qiuli Li.

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