TINY, a Dehydration-responsive Element (DRE)-binding Protein-like Transcription Factor Connecting the DRE- and Ethylene-responsive Element-mediated Signaling Pathways in Arabidopsis

Dehydration-responsive element-binding proteins (DREBs) and ethylene-responsive element (ERE) binding factors are two major subfamilies of the AP2/ethylene-responsive element-binding protein family and play crucial roles in the regulation of abiotic- and biotic-stress responses, respectively. In the present work, we have reported a previously identified DREB-like factor, TINY, that was involved in both abiotic- and biotic-stress signaling pathways. TINY was capable of binding to both DRE and ERE with similar affinity and could activate the expression of reporter genes driven by either of these two elements in tobacco cells. The 15th amino acid in the APETALA2 (AP2)/ethylene-responsive element-binding factor domain was demonstrated to be essential for its specific binding to ERE, whereas the 14th and 19th amino acids were responsible for the binding to DRE. The expression of TINY was greatly activated by drought, cold, ethylene, and slightly by methyl jasmonate. Additionally, overexpression of TINY in Arabidopsis resulted in elevated expressions of both the DRE- and the ERE-containing genes. Moreover, the expression of DRE-regulated genes, such as COR6.6 and ERD10, was up-regulated upon ethylene treatment, and the expression of ERE-regulated genes, such as HLS1, was also increased by cold stress, when the expression of TINY was being induced. These results strongly suggested that TINY might play a role in the cross-talk between abiotic- and biotic-stress-responsive gene expressions by connecting the DRE- and ERE-mediated signaling pathways. The results herein might promote the understanding of the mechanisms of specific DNA recognition and gene expression regulation by DREBs. Dehydration-responsive element-binding proteins (DREBs) and ethylene-responsive element (ERE) binding factors are two major subfamilies of the AP2/ethylene-responsive element-binding protein family and play crucial roles in the regulation of abiotic- and biotic-stress responses, respectively. In the present work, we have reported a previously identified DREB-like factor, TINY, that was involved in both abiotic- and biotic-stress signaling pathways. TINY was capable of binding to both DRE and ERE with similar affinity and could activate the expression of reporter genes driven by either of these two elements in tobacco cells. The 15th amino acid in the APETALA2 (AP2)/ethylene-responsive element-binding factor domain was demonstrated to be essential for its specific binding to ERE, whereas the 14th and 19th amino acids were responsible for the binding to DRE. The expression of TINY was greatly activated by drought, cold, ethylene, and slightly by methyl jasmonate. Additionally, overexpression of TINY in Arabidopsis resulted in elevated expressions of both the DRE- and the ERE-containing genes. Moreover, the expression of DRE-regulated genes, such as COR6.6 and ERD10, was up-regulated upon ethylene treatment, and the expression of ERE-regulated genes, such as HLS1, was also increased by cold stress, when the expression of TINY was being induced. These results strongly suggested that TINY might play a role in the cross-talk between abiotic- and biotic-stress-responsive gene expressions by connecting the DRE- and ERE-mediated signaling pathways. The results herein might promote the understanding of the mechanisms of specific DNA recognition and gene expression regulation by DREBs. With the completion of the Arabidopsis thaliana genome sequence, it is possible to identify and analyze the entire complement of transcription factors in plant. Arabidopsis dedicates ∼5.9% of its estimated total number of genes to code for transcription factors, which is 1.3 times that of Drosophila and 1.7 times that of Caenorhabditis elegans and yeast (1Riechmann J.L. Heard J. Martin G. Reuber L. Jiang C. Keddie J. Adam L. Pineda O. Ratcliffe O.J. Samaha R.R. Creelman R. Pilgrim M. Broun P. Zhang J.Z. Ghandehari D. Sherman B.K. Yu G. Science. 2000; 290: 2105-2110Crossref PubMed Scopus (2104) Google Scholar). APETALA2 (AP2) 2The abbreviations used are:AP2APETALA2ERFethylene-responsive element binding factorEREethylene-responsive elementDREdehydration-responsive elementDREBDRE-binding proteinCBFC-repeat binding factorwDREwide-type DRE element with the sequence 5′-GATATACTACCGACATGAGTTC-3′mDREmutated DRE element with the sequence 5′-GATATACTATTTTCATGAGTTC-3′wEREwide-type ERE element with the sequence 5′-CGCAGACATAGCCGCCATTT-3′mEREmutated ERE element with the sequence 5′-CGCAGACATATCCTCCATTT-3′SDsynthetic dropoutCaMV 35Scauliflower mosaic virus 35SMeJAmethyl jasmonateAVGl-α-(2-aminoethoxyvinyl)-glycineGSTglutathione S-transferase./ethylene-responsive element-binding protein is among the three largest families of transcription factors in Arabidopsis, and this family of transcription factors is plant-specific and contains the highly conserved AP2/ethylene-responsive element binding factor (ERF) DNA-binding domain (1Riechmann J.L. Heard J. Martin G. Reuber L. Jiang C. Keddie J. Adam L. Pineda O. Ratcliffe O.J. Samaha R.R. Creelman R. Pilgrim M. Broun P. Zhang J.Z. Ghandehari D. Sherman B.K. Yu G. Science. 2000; 290: 2105-2110Crossref PubMed Scopus (2104) Google Scholar, 2Riechmann J.L. Meyerowitz E.M. Biol. Chem. 1998; 379: 633-646PubMed Google Scholar). Based on the similarities of the amino acid sequences in the AP2/ERF domain, the members of this family are classified into five subfamilies: AP2, dehydration-responsive element-binding protein (DREB, A1–A6), ERF (B1–B6), related to ABI3/VP1 (RAV), and others (3Sakuma Y. Liu Q. Dubouzet J.G. Abe H. Shinozaki K. Yamaguchi-Shinozaki K. Biochem. Biophys. Res. Commun. 2002; 290: 998-1009Crossref PubMed Scopus (1371) Google Scholar). The DREB and ERF subfamilies have received considerable attention and have been extensively researched over the years due to their participation in plant responses to abiotic and biotic stresses. The DREB subfamily was demonstrated to play a major role in cold-stress and osmotic-stress signal transduction pathways by recognizing the dehydration-responsive element (DRE)/C-repeat with a core sequence of A/GCCGAC (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar, 5Stockinger E.J. Gilmour S.J. Thomashow M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1035-1040Crossref PubMed Scopus (1411) Google Scholar, 6Medina J. Bargues M. Terol J. Perez-Alonso M. Salinas J. Plant Physiol. 1999; 119: 463-470Crossref PubMed Scopus (344) Google Scholar, 7Shinozaki K. Yamaguchi-Shinozaki K. Curr. Opin. Plant Biol. 2000; 3: 217-223Crossref PubMed Google Scholar, 8Nakashima K. Shinwari Z.K. Sakuma Y. Seki M. Miura S. Shinozaki K. Yamaguchi-Shinozaki K. Plant Mol. Biol. 2000; 42: 657-665Crossref PubMed Scopus (339) Google Scholar, 9Dubouzet J.G. Sakuma Y. Ito Y. Kasuga M. Dubouzet E.G. Miura S. Seki M. Shinozaki K. Yamaguchi-Shinozaki K. Plant J. 2003; 33: 751-763Crossref PubMed Scopus (1310) Google Scholar), whereas the ERF subfamily was mainly involved in plant responses to biotic stresses, such as pathogenstress, wounding-stress and ethylene signal, by recognizing the ethylene-responsive element (ERE), also known as the GCC-box, with a core sequence of AGCCGCC (10Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar, 11Hao D. Ohme-Takagi M. Sarai A. J. Biol. Chem. 1998; 273: 26857-26861Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 12Fujimoto S.Y. Ohta M. Usui A. Shinshi H. Ohme-Takagi M. Plant Cell. 2000; 12: 393-404Crossref PubMed Scopus (855) Google Scholar, 13Guo H. Ecker J.R. Curr. Opin. Plant Biol. 2004; 7: 40-49Crossref PubMed Scopus (712) Google Scholar, 14Broekaert W.F. Delaure S.L. Bolle De M.F. Cammue B.P. Annu. Rev. Phytopathol. 2006; 44: 393-416Crossref PubMed Scopus (332) Google Scholar). However, recent works have revealed some new characteristics of these two subfamily members. For example, ABA-insensitive 4, the only member of the A-3 group, was recently reported to regulate the nuclear gene expression in the plant response to plastid-to-nucleus retrograde signals through binding a conserved motif of CCAC in the promoter of a retrograde-regulated gene (15Koussevitzky S. Nott A. Mockler T.C. Hong F. Sachetto-Martins G. Surpin M. Lim J. Mittler R. Chory J. Science. 2007; 316: 715-719Crossref PubMed Google Scholar). In addition, Sasaki et al. isolated two B-4 group genes from tobacco, named wound-responsive AP2/ERF-like factors 1 and 2, and indicated that they were positive regulators for wound-induced expression of a tobacco peroxidase gene through specifically interacting with the vascular system-specific and wound-responsive cis-element (16Sasaki K. Mitsuhara I. Seo S. Ito H. Matsui H. Ohashi Y. Plant J. 2007; 50: 1079-1092Crossref PubMed Scopus (36) Google Scholar). These findings suggest that the DREB and ERF proteins play multiple roles during the course of plant growth and that the differences in their DNA-binding specificities are accompanied by the functional diversity. APETALA2 ethylene-responsive element binding factor ethylene-responsive element dehydration-responsive element DRE-binding protein C-repeat binding factor wide-type DRE element with the sequence 5′-GATATACTACCGACATGAGTTC-3′ mutated DRE element with the sequence 5′-GATATACTATTTTCATGAGTTC-3′ wide-type ERE element with the sequence 5′-CGCAGACATAGCCGCCATTT-3′ mutated ERE element with the sequence 5′-CGCAGACATATCCTCCATTT-3′ synthetic dropout cauliflower mosaic virus 35S methyl jasmonate l-α-(2-aminoethoxyvinyl)-glycine glutathione S-transferase. The AP2/ERF domains of the DREB and ERF proteins are closely related to each other, but their target DNA-binding sites are different. The solution structure of AtERF1 in complex with ERE revealed that seven amino acids in the AP2/ERF domain made direct contact with DNA (17Allen M.D. Yamasaki K. Ohme-Takagi M. Tateno M. Suzuki M. EMBO J. 1998; 17: 5484-5496Crossref PubMed Scopus (403) Google Scholar). Interestingly, these amino acids were completely conserved in all reported DREBs and ERFs except Trp10, which was also highly conserved (18Hao D. Yamasaki K. Sarai A. Ohme-Takagi M. Biochemistry. 2002; 41: 4202-4208Crossref PubMed Scopus (90) Google Scholar). Thus, the determinants of the different DNA-binding specificity were those divergent residues in the AP2/ERF domain between DREBs and ERFs. Two conserved amino acids in the AP2/ERF domain differ between them: the 14th Val and the 19th Glu in the DREBs and Ala and Asp in the corresponding positions of the ERFs. Previous studies have shown that the Val-14 and Glu-19, especially Val-14, were essential for specific binding to DRE (3Sakuma Y. Liu Q. Dubouzet J.G. Abe H. Shinozaki K. Yamaguchi-Shinozaki K. Biochem. Biophys. Res. Commun. 2002; 290: 998-1009Crossref PubMed Scopus (1371) Google Scholar, 18Hao D. Yamasaki K. Sarai A. Ohme-Takagi M. Biochemistry. 2002; 41: 4202-4208Crossref PubMed Scopus (90) Google Scholar, 19Cao Z.F. Li J. Chen F. Li Y.Q. Zhou H.M. Liu Q. Biochemistry (Mosc.). 2001; 66: 623-627Crossref PubMed Scopus (52) Google Scholar, 20Qin F. Sakuma Y. Li J. Liu Q. Li Y.Q. Shinozaki K. Yamaguchi-Shinozaki K. Plant Cell Physiol. 2004; 45: 1042-1052Crossref PubMed Scopus (309) Google Scholar). In addition, Ala-37 was reported to play a key role in binding to both DRE and ERE (21Liu Y. Zhao T.J. Liu J.M. Liu W.Q. Liu Q. Yan Y.B. Zhou H.M. FEBS Lett. 2006; 580: 1303-1308Crossref PubMed Scopus (78) Google Scholar). However, there is no report about the amino acids that are crucial in the specific recognition of ERE by far. TINY gene was first isolated through a transposon-mutagenesis experiment designed to recover dominant gain-of-function alleles in Arabidopsis (22Wilson K. Long D. Swinburne J. Coupland G. Plant Cell. 1996; 8: 659-671Crossref PubMed Scopus (213) Google Scholar) and then identified as a DREB-like transcription factor binding to DRE (23Yu J. Sun S. Jiang Y. Ma X. Chen F. Zhang G. Fang X. Polymer. 2006; 47: 2533-2538Crossref Scopus (17) Google Scholar, 24Zhifang C. Feng C. Jie L. Yiqin L. Haimeng Z. Guiyou Z. Qiang L. Tsinghua Sci. Technol. 2001; 6: 432-437Google Scholar) and classified into the A-4 group (3Sakuma Y. Liu Q. Dubouzet J.G. Abe H. Shinozaki K. Yamaguchi-Shinozaki K. Biochem. Biophys. Res. Commun. 2002; 290: 998-1009Crossref PubMed Scopus (1371) Google Scholar), which suggested that, like other DREB proteins, TINY might play a role in response to abiotic stresses. Besides, enhancement of the expression of TINY gene caused a partial constitutive triple response, such as the short, thick hypocotyl and short root of the 3-day-old dark-grown seedling, which implied that TINY might be involved in ethylene response (22Wilson K. Long D. Swinburne J. Coupland G. Plant Cell. 1996; 8: 659-671Crossref PubMed Scopus (213) Google Scholar). Here, detailed work was carried out to explore the function of TINY in plants. We analyzed its stress-inducible expression profile, the DNA-binding and trans-activation properties, and the effect of overexpressing TINY in Arabidopsis plants. TINY was induced by not only abiotic stresses, but also the defense signal molecules ethylene and methyl jasmonate (MeJA). Interestingly, TINY could bind to both DRE and ERE with similar affinity to further activate the expression of downstream genes driven by either of these two cis-elements, suggesting that TINY might play a role in the cross-talk between the DRE- and ERE-mediated signaling pathways. Additionally, we found that Ser-15 in the AP2/ERF domain was essential for specific binding to ERE of TINY. The results herein may improve our understanding of the roles of AP2/ethylene-responsive element-binding protein transcription factors in plant signal transduction pathways. Plant Materials and Stress Treatment—Plants (Arabidopsis thaliana ecotype Columbia) were grown on germination medium agar plates as described previously (25Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1994; 6: 251-264Crossref PubMed Scopus (1571) Google Scholar) for 3 weeks and subjected to stress treatments. For treatment with water, salts, and ethylene, plants were grown hydroponically in dH2O, 250 mm NaCl, or 1 mm ethephon solution. For cold stress treatment, the plants were kept in a 4 °C refrigerator. For drought stress treatment, the plants were placed on filter paper on a clean bench under dim light. For treatment with MeJA, 4-week-old plants grown in soil were sprayed with 50 μm MeJA solution. MeJA was dissolved in 50% ethanol as a 10 mm stock solution. The MeJA stock solution was diluted to 50 μm with water. In each case, the plants were subjected to the stress treatment for designated periods and frozen in liquid nitrogen. For AgNO3 (Sigma) and l-α-(2-aminoethoxyvinyl)glycine (AVG, Sigma) treatment, 19-day-old plants were transferred to plates containing 10 μm AgNO3 or 20 μm AVG, and assays were performed after additional 2 days of growth. Site-directed Mutagenesis—The TINY gene (22Wilson K. Long D. Swinburne J. Coupland G. Plant Cell. 1996; 8: 659-671Crossref PubMed Scopus (213) Google Scholar) was kindly provided by Dr. George Coupland (Max Planck Institute for Plant Breeding, Cologne, Germany). The 14th and 19th residues in TINY were singly or doubly replaced by alanine and aspartic acid named T_V14A, T_E19D, and T_V14AE19D, respectively. The point mutation in 15th amino acid residue of TINY was obtained by singly replaced serine by cysteine and named T_S15C. All mutants were generated by PCR-mediated overlapping, and the mutants were sequenced and cloned into pGADT7 for yeast one-hybrid assay or pGEX-4T-1 for GST fusion protein preparation. In Vivo DNA-binding Experiment Using the Yeast One-hybrid System—Construction of DRE reporter plasmids and selection of the yeast reporter system were performed as described previously (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar). Construction of ERE reporter plasmids used the same method. A 70-bp region containing two ERE sequences from the Arabidopsis HLS1 gene (26Lehman A. Black R. Ecker J.R. Cell. 1996; 85: 183-194Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) (5′-TAATAATGAGTTAACGCAGACATAGCCGCCATTTTTAATAATGAGTTAACGCAGACATAGCCGCCATTTT-3′, the core sequence of ERE element is underlined) was synthesized and ligated into two tandemly repeated copies, and then inserted into the SmaI site of the multiple cloning site upstream of the HIS3 minimal promoter in the pHISi-1 expression vector (Clontech, Palo Alto, CA) and the LacZ minimal promoter in the pLacZi expression vector (Clontech), respectively. The wERE reporter system was constructed by simultaneously transforming these two plasmids into yeast strain YM4271. The reporter yeast containing the mERE with the substitution of AGCCGCC with ATCCTCC was constructed by using the same method. The full-length coding regions of TINY and its mutants were cloned into the EcoRI and SalI sites of the pGADT7 vector (Clontech). The constructs were transformed into the reporter yeast cells as described previously (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar). The growth status of the transformed yeast cells was compared on synthetic dextrose (SD) medium without His, Ura, and Leu (SD/-His-/Ura/-Leu) with 0 mm, 30 mm, or 50 mm 3-aminotriazole (a competitive inhibitor of the HIS3 gene product). The colony-lift filter assay used to measure β-galactosidase activity was performed to test the expression of the LacZ reporter gene as described in the Yeast Protocols Handbook (Clontech). GST Fusion Protein Preparation and Gel Mobility Shift Assays—The 363-bp (1–363) fragments of TINY and its mutants containing the DNA-binding domain were prepared by the primer pairs: 5′-AAAAGAATTCATGATAGCTTCAGAGAGTAC-3′ (forward), 5′-AAAAGTCGACTTAGGTCTCCATGTGTGCGGCTTTG-3′ (reverse). Each of the fragments was cloned into the EcoRI-SalI sites of the pGEX-4T-1 vector (Amersham Biosciences), and the constructs were transformed into Escherichia coli strain BL21(DE3) to produce the GST fusion protein. The GST fusion protein was separated using a glutathione-Sepharose 4B column (Amersham Biosciences) according to the manufacturer's instructions. Gel mobility shift assays were performed as described previously (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar). The 71-bp DNA fragment containing wild-type or mutated DRE sequence and 29-bp DNA fragment containing wild-type or mutated ERE sequence (Fig. 2A) were labeled by filling in 5′ overhangs with [32P]dCTP and the Klenow fragment, respectively. The DNA-binding reaction was constructed as described previously (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar). For competition experiments, unlabeled competitors were incubated with the protein at 25 °C for 5 min before the addition of labeled probes, which were further incubated for 30 min (9Dubouzet J.G. Sakuma Y. Ito Y. Kasuga M. Dubouzet E.G. Miura S. Seki M. Shinozaki K. Yamaguchi-Shinozaki K. Plant J. 2003; 33: 751-763Crossref PubMed Scopus (1310) Google Scholar). Single Molecule Force Measurement Using AFM—The DNA sequences used in all force measurements were custom synthesized from SBS Genetech Co. Ltd. (Beijing, China). These include the DRE element sequence, 5′-NH2-GATATACTACCGACATGAGTTC-3′, and its complementary single strand DNA, 3′-CTATATGATGGCTGTACTCAAG-5′; the ERE element sequence, 5′-NH2-CGCAGACATAGCCGCCATTT-3′, and its complementary single strand DNA, 3′-GCGTCTGTATCGGCGGTAAA-5′ (the element sequences are underlined). Chemical modification of the AFM tips and substrates and force measurements were performed according to previously reported procedures (23Yu J. Sun S. Jiang Y. Ma X. Chen F. Zhang G. Fang X. Polymer. 2006; 47: 2533-2538Crossref Scopus (17) Google Scholar). Trans-activation Activity of the DRE- and ERE-LUC Reporter Genes in Plant Cells—The trans-activation activity of TINY was measured in a constructed dual reporter system (Fig. 4A) as described previously (21Liu Y. Zhao T.J. Liu J.M. Liu W.Q. Liu Q. Yan Y.B. Zhou H.M. FEBS Lett. 2006; 580: 1303-1308Crossref PubMed Scopus (78) Google Scholar). The DRE dual reporter system was established by our previous work (27Zhao T.J. Sun S. Liu Y. Liu J.M. Liu Q. Yan Y.B. Zhou H.M. J. Biol. Chem. 2006; 281: 10752-10759Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The ERE reporter construct containing a 4×HLS GCC-box (12Fujimoto S.Y. Ohta M. Usui A. Shinshi H. Ohme-Takagi M. Plant Cell. 2000; 12: 393-404Crossref PubMed Scopus (855) Google Scholar) was the generous gift from Dr. Ohme-Takagi (Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, Japan). For effector plasmids, theβ-glucuronidase (GUS) gene in pBI221 (Clontech) was replaced by the coding region of TINY. The tobacco mosaic virus Ω sequence (28Gallie D.R. Sleat D.E. Watts J.W. Turner P.C. Wilson T.M. Nucleic Acids Res. 1987; 15: 8693-8711Crossref PubMed Scopus (164) Google Scholar) was inserted downstream of the CaMV 35S promoter in pBI221 to enhance the efficiency of translation. The effector plasmid consisting of TINY and either DRE reporter plasmid or ERE reporter plasmid were delivered into the protoplasts. The plasmid carrying the CaMV 35S promoter-Renilla luciferase gene (R reporter plasmid) was co-transfected with both reporter and effector plasmids as an internal control. The isolation of tobacco protoplasts from the BY2 suspension cultures and the polyethylene glycol-mediated DNA transformation of tobacco protoplasts were performed as described previously (21Liu Y. Zhao T.J. Liu J.M. Liu W.Q. Liu Q. Yan Y.B. Zhou H.M. FEBS Lett. 2006; 580: 1303-1308Crossref PubMed Scopus (78) Google Scholar, 29Abeles F.B. Morgan P.W. Saltveit Jr., M.E. Ethylene in Plant Biology. Academic Press, San Diego, CA1992: 196Google Scholar). The Renilla and firefly luciferase activities were measured according to the manufacturer's instructions (Promega, Dual-Luciferase Reporter Assay System). Trans-activation Activity Analysis in Yeast Cells—A yeast one-hybrid system derived from the GAL4 two-hybrid system (Fig. 4C) was developed to test whether the C-terminal of TINY protein functions as a transcriptional activation domain. The effector plasmids were constructed by inserting the 333-bp (322–654) fragment of TINY or the full-length coding region of DREB1A (a positive control) downstream of the yeast GAL4 DNA-binding domain of pGBKT7 (Clontech). The fusion plasmids and the vector as a negative control were transformed into yeast strain AH109 (Clontech). The transformants were analyzed on the synthetic dextrose medium without Trp, His, and Ade (SD/-Trp/-His/-Ade) to test the expression of the HIS3 and ADE2 reporter genes. The colony-lift filter assay used to measure β-galactosidase activity was performed subsequently to test the expression of the LacZ reporter gene. Quantitative Real-time PCR Analysis—Total RNA was prepared by TRIzol Reagent (Invitrogen) as instructed. cDNA was synthesized by using Moloney murine leukemia virus reverse transcriptase (Promega) with the oligo(dT)15 primer according to the manufacturer's instructions. Quantitative real-time PCR using SYBR Green I Dye (Bio-V) was performed on Mx3000PTM (Stratagene, La Jolla, CA). Three replicate PCR amplifications were performed for each sample. The Actin2 gene was also amplified as an internal control (30An Y.Q. McDowell J.M. Huang S. McKinney E.C. Chambliss S. Meagher R.B. Plant J. 1996; 10: 107-121Crossref PubMed Scopus (408) Google Scholar). The amount of the transcripts of each gene, normalized to the internal reference Actin2, is analyzed using 2-ΔΔCt method (31Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar). The amount of the transcripts of each target gene under normal condition, or in control transgenic plants (vec), was designated as 1.0. The primers used for the real-time PCR were: TINY, 5′-CACAGTCTTCTTCTTCGCTAGAGTC-3′ (forward), 5′-GTGATAACGAGGCAGGAATCAT-3′ (reverse); Actin2, 5′-GACCTTTAACTCTCCCGCTATGTA-3′ (forward), 5′-GTGGTGAACATGTAACCTCTCTCTG-3′ (reverse); COR6.6, 5′-AGTATATCGGATGCGGCAGT-3′ (forward), 5′-CAAACGTAGTACATCTAAAGGGAGA-3′ (reverse); COR15A, 5′-AAAACTCAGTTCGTCGTCGTTT-3′ (forward), 5′-GCTTCTTTACCCAATGTATCTGC-3′ (reverse); COR78, 5′-CAAAACAGAGCACTTACACAGAGAA-3′ (forward), 5′-CATAATCTCTACCCGACACACTTTT-3′ (reverse); ERD10, 5′-AAGGGATTTATGGACAAGATCAAA-3′ (forward), 5′-CACAAACTTGGAGAACAGCTAGAA-3′ (reverse); PDF1.2 (32Brown R.L. Kazan K. McGrath K.C. Maclean D.J. Manners J.M. Plant Physiol. 2003; 132: 1020-1032Crossref PubMed Scopus (338) Google Scholar), 5′-TTGCTGCTTTCGACGCA-3′ (forward), 5′-TGTCCCACTTGGCTTCTCG-3′ (reverse); and HLS1, 5′-TCGAATATCCACCCGAGTCATG-3′ (forward), 5′-CTTCTCCTCCGATTCCATACATAA-3′ (reverse). Plant Transformation—The plasmid used for the transformation of Arabidopsis was constructed with the coding region of the TINY cDNA. The coding region fragment was cloned into a multicloning site of the pBI121 vector (Clontech) (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar). The plasmid and the vector as the transgenic control were then introduced into Agrobacterium tumefaciens strain EHA105. Plants were transformed using a vacuum infiltration method as described previously (4Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2390) Google Scholar). TINY Can Bind to Both DRE and ERE Elements with Similar Affinity—To analyze the DNA-binding property of TINY, the entire coding region of TINY was fused in-frame with the GAL4 activation domain in a pGADT7 vector and subjected to yeast one-hybrid assay using four reporter yeast strains, which carried the dual reporter gene HIS3 and LacZ under the control of wild-type DRE, mutated DRE, wild-type ERE, and mutated ERE sequences, respectively. The growth status of these transformed yeast cells was analyzed. As shown in Fig. 1A (upper panel), in wild-type DRE reporter yeasts, the transformants carrying the recombinant plasmid TINY/pGADT7 and DREB1A(CBF3)/pGADT7 (a positive control) could grow well on SD/-His/-Ura/-Leu containing 30 mm 3-aminotriazole, and show the expression of LacZ activity, whereas the transformants with the vector plasmid pGADT7, as a negative control, could neither grow on SD/-His/-Ura/-Leu nor show the expression of lacZ. To make a further confirmation, TINY/pGADT7 was transformed into mutated DRE reporter yeasts, and the transformants could not grow on SD/-His/-Ura/-Leu, which indicated that TINY could specifically bind to wild-type DRE in vivo. Similar results were obtained from the ERE reporter system (Fig. 1A, lower panel), showing that TINY could also specifically bind to wild-type ERE. Then the results here indicated that TINY could specifically bind to both DRE and ERE elements. To further confirm the results above, a gel mobility shift assay was carried out to test the binding affinity and specificity of TINY to these two cis-elements in vitro. The 121 amino acids of the DNA-binding domain of TINY were expressed in E. coli as a fusion protein with GST and was used for gel mobility shift assay with 32P-labeled probes, WD and M1–M5 (Fig. 1B). As shown in Fig. 1C, the recombinant protein bound the wild-type 71-bp fragment containing DRE element, but not the base-substituted 71-bp fragments M1, M2, and M3, in which the DRE element was mutated. By contrast, the protein still bound fragments M4 and M5, which still contained the DRE element. Therefore, TINY could specifically bind to the DRE element. Likewise, the TINY fusion protein bound the wild-type 29-bp fragment-containing ERE element, but not the base-substituted fragment containing mutated ERE element (Fig. 1C). As the negative control, no retardation band was detected when the pure GST protein was tested using any of these probes (Fig. 1C). These results indicate that TINY could specifically interact with both DRE and ERE elements. The DNA-binding specificity of TINY was further confirmed by the competition experiments. The specific interaction between TINY and DRE element was effectively competed by either unlabeled DRE or ERE, and competitive abilities of DRE and ERE were almost the same. Similarly, unlabeled DRE and ERE showed almost the same abilities to compete with the labeled ERE to bind to TINY (Fig. 1D). These observations clearly show that TINY interacted specifically with both the DRE and ERE sequences with similar binding affinity. To make a quantitative analysis of the binding affinity of TINY with DRE and ERE, we used AFM to quantify the binding strength, according to our previously established system (23Yu J. Sun S. Jiang Y. Ma X. Chen F. Zhang G. Fang X. Polymer. 2006; 47: 2533-2538Crossref Scopus (17) Google Scholar). By fitting a Gaussian distribution to the single peak in the force distribution histogram, the most probable force, 91.3 ± 6.1 piconewtons, for the specific single molecular interaction force of TINY-GST/ERE was calculated (Fig. 3B, left panel). This value was similar to that of the single molecule force between TINY and DRE obtained under the same experiment conditions before, which was 83.5 ± 3.4 piconewtons (23Yu J. Sun S. Jiang Y. Ma X. Chen F. Zhang G. Fang X. Polymer. 2006; 47: 2533-2538Crossref Scopus (17) Google Scholar). The results he