Plant Mol Biol (2014) 86:609–625 DOI 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
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 transcriptional activator, and could bind to the TACA(A/G) T sequence. AtDi19-3 expression in plants was remarkably 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 greening in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression 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-tolerance, 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, China e-mail: [email protected] Present Address: L.-X. Qin Institute of Cotton, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, China
exhibited more drought-sensitivity than wild type. Furthermore, expression of the genes related to ABA signaling 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 expression of specific sets of genes (Shinozaki et al. 2003). Several signal transduction pathways exist in plants responding to abiotic stresses, including calcium-independent mitogen-activated protein kinase (MAPK) cascade signaling, calcium-dependent protein kinases (CDPK) phosphorylated 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 germination and seedling growth, but loss-of-function mutations 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+
13
610
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 germination, 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 positive regulators, have been identified in plants (Himmelbach et al. 2003; Israelsson et al. 2006). In ABA-dependent pathway, MYC and MYB recognition sequences are essential for the ABA- and drought-responsive expression of rd22 (Abe et al. 1997). The ABA-responsive regulatory 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 factors (TFs), such as ABA-responsive element binding proteins (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-deficient aba2 mutant (Fujita et al. 2004). Moreover, genetic evidence suggested that stress-signaling pathways for the activation of LEA-like genes which are completely independent of ABA may not exist (Xiong et al. 2002). Therefore, both ABA-dependent and ABA-independent signal transduction pathways may interact and converge to activate 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/His2type zinc finger domain containing two cysteines and two histidines is one of the best-characterized and important
13
Plant Mol Biol (2014) 86:609–625
DNA-binding motifs involved in protein-DNA interaction 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 consensus 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 stability (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 zincfinger proteins have been found to play important roles in plant response to abiotic stresses. For example, Arabidopsis 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 overexpressing STZ, as a transcription repressor, showed growth retardation and increased tolerance to drought stress (Sakamoto et al. 2004). Soybean SCOF-1 enhanced cold tolerance 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/His2-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 tolerance) 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 evolutionarily 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 hydrophilic 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 cotton Di19 proteins, named GhDi19-1 and GhDi19-2, are involved in plant response to salt stress and ABA signaling. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis 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 (pathogenesis-related 1), PR2 and PR5 genes (Liu et al. 2013). In
Plant Mol Biol (2014) 86:609–625
this study, we report that AtDi19-3 as a transcriptional activator 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 exogenous ABA, but sensitive to H2O2 during seed germination and early seedling development, while AtDi19-3 overexpression transgenic plants display the opposite phenotype.
611
Materials and methods
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 flashfreezing 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′.
Plants materials and growth conditions
EMSA assay
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, followed by washing with sterile water. The sterilized Arabidopsis 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.
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-binding 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 maltose (NEW ENGLAND) was used in protein/DNA binding analysis, using MBP protein as control. A pair of 30 bp oligonuleotides (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.
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 introduced into Arabidopsis by the floral dip method (Clough and Bent 1998). The harvested seeds were germinated on selective MS medium for selecting transgenic plants. Subsequently, 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 construction 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
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 transgenic Arabidopsis was conducted according to a modified protocol (Xu et al. 2013). Seedlings (5- and 10-dayold) and mature leaves were collected for assaying GUS expression. The samples were stained at 37 °C 6-8 h in
13
612
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 cultured 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 controls. GUS staining patterns were confirmed by observing at least four different transgenic lines. The representative 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 complementation 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), respectively. Primers used in the vector construction are: AtDi193 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 transgenic 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 seedlings of wild type, 35S:AtDi19-3-oe, Atdi19-3 + proAtDi193: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, respectively (22 °C, 16 h light/8 h dark) in a plant growth incubator. Seeds were considered successfully germinated when radicals completely penetrated the seed coats. Germination
13
Plant Mol Biol (2014) 86:609–625
rate and proportion of seedlings with opened green cotyledons 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 germinated 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 position. 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 experiments were performed with three technical replications, each line containing at least 100 seeds for analyzing the seed germination 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 seedlings 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 independent 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 mixture 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 independent 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 withheld 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-responsive genes, qRT-PCR analysis was performed with the RNA samples isolated from 10-day-old seedlings treated with or
Plant Mol Biol (2014) 86:609–625
613
Fig. 1 Subcellular localization and transcriptional activation analysis of AtDi19-3 protein. a Nuclear localization of AtDi19-3 protein. Micrographs of root cells of 35S:AtDi19-3-GFP transgenic Arabidopsis. 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 transcriptional 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
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) forward 5′-GGCTTGAAGAAAGTGAGTCTCG-3′ and reverse 5′-GCTACATAGTTCGGAGTTCCACA-3′. Expression levels of Arabidopsis ACTIN2 were monitored with forward 5′-GAAATCACAGCACTTGCACC-3′ and reverse 5′-AAGC CTTTGATCTTGAGAGC-3′ primers to serve as a normalization control. The expression of these genes was analyzed by quantitative RT-PCR using the fluorescent intercalating dye SYBRGreen in a detection system (Opticon2; MJ Research) as described previously (Li et al. 2005). For all the above quantitative 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 accumulated mainly in the nuclei of root cells of the transgenic seedlings, demonstrating that AtDi19-3 is a nuclear-localized 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 positives. The yeast transformants were examined for their growth on selection medium (SD/-Trp-His or SD/-TrpAde) 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
13
614
Plant Mol Biol (2014) 86:609–625
BD-AtDi19-3 vectors grew well. On double nutritiondeficient 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 transcription 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 ciselement, 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 MBPAtDi19-3/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/webtool s/plantcare/html/) revealed it contains ABRE and MBS cisacting elements involved in response to ABA and drought stress. To determine whether AtDi19-3 promoter is inducible in plants under salt, drought and ABA treatments, AtDi19-3 promoter:GUS fusion expression vector was constructed 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 further development, weaker GUS staining was still observed in the mature leaf tips of AtDi19-3 promoter:GUS transgenic plants (Fig. 3b). We further assayed GUS activity in the transgenic plants under salt, ABA and drought treatments. The experimental results revealed that GUS activity 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)
13
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
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 cotyledons 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 transgenic 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
Plant Mol Biol (2014) 86:609–625
615
Fig. 3 Histochemical assay of GUS activity under the control of AtDi19-3 promoter in transgenic Arabidopsis. a A five-day-old seedling. 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 mannitol (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 treatments, 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 60 seedlings per each 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
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
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 transcriptional activator, and could bind to the TACA(A/G) T sequence. AtDi19-3 expression in plants was remarkably 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 greening in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression 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-tolerance, 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, China e-mail: [email protected] Present Address: L.-X. Qin Institute of Cotton, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, China
exhibited more drought-sensitivity than wild type. Furthermore, expression of the genes related to ABA signaling 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 expression of specific sets of genes (Shinozaki et al. 2003). Several signal transduction pathways exist in plants responding to abiotic stresses, including calcium-independent mitogen-activated protein kinase (MAPK) cascade signaling, calcium-dependent protein kinases (CDPK) phosphorylated 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 germination and seedling growth, but loss-of-function mutations 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+
13
610
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 germination, 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 positive regulators, have been identified in plants (Himmelbach et al. 2003; Israelsson et al. 2006). In ABA-dependent pathway, MYC and MYB recognition sequences are essential for the ABA- and drought-responsive expression of rd22 (Abe et al. 1997). The ABA-responsive regulatory 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 factors (TFs), such as ABA-responsive element binding proteins (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-deficient aba2 mutant (Fujita et al. 2004). Moreover, genetic evidence suggested that stress-signaling pathways for the activation of LEA-like genes which are completely independent of ABA may not exist (Xiong et al. 2002). Therefore, both ABA-dependent and ABA-independent signal transduction pathways may interact and converge to activate 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/His2type zinc finger domain containing two cysteines and two histidines is one of the best-characterized and important
13
Plant Mol Biol (2014) 86:609–625
DNA-binding motifs involved in protein-DNA interaction 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 consensus 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 stability (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 zincfinger proteins have been found to play important roles in plant response to abiotic stresses. For example, Arabidopsis 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 overexpressing STZ, as a transcription repressor, showed growth retardation and increased tolerance to drought stress (Sakamoto et al. 2004). Soybean SCOF-1 enhanced cold tolerance 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/His2-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 tolerance) 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 evolutionarily 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 hydrophilic 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 cotton Di19 proteins, named GhDi19-1 and GhDi19-2, are involved in plant response to salt stress and ABA signaling. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis 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 (pathogenesis-related 1), PR2 and PR5 genes (Liu et al. 2013). In
Plant Mol Biol (2014) 86:609–625
this study, we report that AtDi19-3 as a transcriptional activator 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 exogenous ABA, but sensitive to H2O2 during seed germination and early seedling development, while AtDi19-3 overexpression transgenic plants display the opposite phenotype.
611
Materials and methods
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 flashfreezing 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′.
Plants materials and growth conditions
EMSA assay
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, followed by washing with sterile water. The sterilized Arabidopsis 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.
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-binding 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 maltose (NEW ENGLAND) was used in protein/DNA binding analysis, using MBP protein as control. A pair of 30 bp oligonuleotides (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.
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 introduced into Arabidopsis by the floral dip method (Clough and Bent 1998). The harvested seeds were germinated on selective MS medium for selecting transgenic plants. Subsequently, 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 construction 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
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 transgenic Arabidopsis was conducted according to a modified protocol (Xu et al. 2013). Seedlings (5- and 10-dayold) and mature leaves were collected for assaying GUS expression. The samples were stained at 37 °C 6-8 h in
13
612
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 cultured 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 controls. GUS staining patterns were confirmed by observing at least four different transgenic lines. The representative 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 complementation 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), respectively. Primers used in the vector construction are: AtDi193 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 transgenic 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 seedlings of wild type, 35S:AtDi19-3-oe, Atdi19-3 + proAtDi193: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, respectively (22 °C, 16 h light/8 h dark) in a plant growth incubator. Seeds were considered successfully germinated when radicals completely penetrated the seed coats. Germination
13
Plant Mol Biol (2014) 86:609–625
rate and proportion of seedlings with opened green cotyledons 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 germinated 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 position. 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 experiments were performed with three technical replications, each line containing at least 100 seeds for analyzing the seed germination 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 seedlings 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 independent 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 mixture 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 independent 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 withheld 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-responsive genes, qRT-PCR analysis was performed with the RNA samples isolated from 10-day-old seedlings treated with or
Plant Mol Biol (2014) 86:609–625
613
Fig. 1 Subcellular localization and transcriptional activation analysis of AtDi19-3 protein. a Nuclear localization of AtDi19-3 protein. Micrographs of root cells of 35S:AtDi19-3-GFP transgenic Arabidopsis. 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 transcriptional 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
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) forward 5′-GGCTTGAAGAAAGTGAGTCTCG-3′ and reverse 5′-GCTACATAGTTCGGAGTTCCACA-3′. Expression levels of Arabidopsis ACTIN2 were monitored with forward 5′-GAAATCACAGCACTTGCACC-3′ and reverse 5′-AAGC CTTTGATCTTGAGAGC-3′ primers to serve as a normalization control. The expression of these genes was analyzed by quantitative RT-PCR using the fluorescent intercalating dye SYBRGreen in a detection system (Opticon2; MJ Research) as described previously (Li et al. 2005). For all the above quantitative 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 accumulated mainly in the nuclei of root cells of the transgenic seedlings, demonstrating that AtDi19-3 is a nuclear-localized 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 positives. The yeast transformants were examined for their growth on selection medium (SD/-Trp-His or SD/-TrpAde) 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
13
614
Plant Mol Biol (2014) 86:609–625
BD-AtDi19-3 vectors grew well. On double nutritiondeficient 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 transcription 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 ciselement, 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 MBPAtDi19-3/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/webtool s/plantcare/html/) revealed it contains ABRE and MBS cisacting elements involved in response to ABA and drought stress. To determine whether AtDi19-3 promoter is inducible in plants under salt, drought and ABA treatments, AtDi19-3 promoter:GUS fusion expression vector was constructed 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 further development, weaker GUS staining was still observed in the mature leaf tips of AtDi19-3 promoter:GUS transgenic plants (Fig. 3b). We further assayed GUS activity in the transgenic plants under salt, ABA and drought treatments. The experimental results revealed that GUS activity 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)
13
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
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 cotyledons 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 transgenic 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
Plant Mol Biol (2014) 86:609–625
615
Fig. 3 Histochemical assay of GUS activity under the control of AtDi19-3 promoter in transgenic Arabidopsis. a A five-day-old seedling. 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 mannitol (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 treatments, 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 60 seedlings per each 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
