ABFs, a Family of ABA-responsive Element Binding Factors
2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês
10.1074/jbc.275.3.1723
ISSN1083-351X
AutoresHyung-in Choi, Jung-Hee Hong, Jin-ok Ha, Jung‐youn Kang, Soo Young Kim,
Tópico(s)Plant nutrient uptake and metabolism
ResumoAbscisic acid (ABA) plays an important role in environmental stress responses of higher plants during vegetative growth. One of the ABA-mediated responses is the induced expression of a large number of genes, which is mediated bycis-regulatory elements known as abscisic acid-responsive elements (ABREs). Although a number of ABRE binding transcription factors have been known, they are not specifically from vegetative tissues under induced conditions. Considering the tissue specificity of ABA signaling pathways, factors mediating ABA-dependent stress responses during vegetative growth phase may thus have been unidentified so far. Here, we report a family of ABRE binding factors isolated from young Arabidopsis plants under stress conditions. The factors, isolated by a yeast one-hybrid system using a prototypical ABRE and named as ABFs (ABREbinding factors) belong to a distinct subfamily of bZIP proteins. Binding site selection assay performed with one ABF showed that its preferred binding site is the strong ABRE, CACGTGGC. ABFs can transactivate an ABRE-containing reporter gene in yeast. Expression of ABFs is induced by ABA and various stress treatments, whereas their induction patterns are different from one another. Thus, a new family of ABRE binding factors indeed exists that have the potential to activate a large number of ABA/stress-responsive genes inArabidopsis. Abscisic acid (ABA) plays an important role in environmental stress responses of higher plants during vegetative growth. One of the ABA-mediated responses is the induced expression of a large number of genes, which is mediated bycis-regulatory elements known as abscisic acid-responsive elements (ABREs). Although a number of ABRE binding transcription factors have been known, they are not specifically from vegetative tissues under induced conditions. Considering the tissue specificity of ABA signaling pathways, factors mediating ABA-dependent stress responses during vegetative growth phase may thus have been unidentified so far. Here, we report a family of ABRE binding factors isolated from young Arabidopsis plants under stress conditions. The factors, isolated by a yeast one-hybrid system using a prototypical ABRE and named as ABFs (ABREbinding factors) belong to a distinct subfamily of bZIP proteins. Binding site selection assay performed with one ABF showed that its preferred binding site is the strong ABRE, CACGTGGC. ABFs can transactivate an ABRE-containing reporter gene in yeast. Expression of ABFs is induced by ABA and various stress treatments, whereas their induction patterns are different from one another. Thus, a new family of ABRE binding factors indeed exists that have the potential to activate a large number of ABA/stress-responsive genes inArabidopsis. abscisic acid abscisic acid-responsive element basic leucine zipper polymerase chain reaction coupled reverse transcription and PCR glutathione S-transferase electrophoretic mobility shift assay open reading frame Dc3 promoter binding factors Abscisic acid (ABA)1 is one of the major plant hormones that plays an important role during plant growth and development (1.Zeevaart J.A.D. Creelman R.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1988; 39: 439-473Crossref Google Scholar, 2.Leung J. Giraudat J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 199-222Crossref PubMed Scopus (997) Google Scholar). The hormone controls several physiological processes during seed development and germination. During vegetative growth, ABA mediates responses to various adverse environmental conditions such as drought, high salt, and cold/freezing (3.Shinozaki K. Yamaguchi-Shinozaki K. Curr. Opin. Biotechnol. 1996; 7: 161-167Crossref PubMed Scopus (397) Google Scholar, 4.Thomashow M.F. Plant Physiol. 1998; 118: 1-7Crossref PubMed Scopus (548) Google Scholar). The ABA-mediated adaptive responses to environmental stresses include stomatal closure and expression of a large number of genes involved in stress tolerance (5.Ingram J. Bartels D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1777) Google Scholar). These and other ABA-mediated stress responses are critical to plant survival and productivity. Hence, ABA biosynthetic mutants are prone to wilting and cannot grow well even under normal, unstressed conditions. Many cis-elements known as ABA-responsive elements (ABREs) have been identified from the promoter analysis of ABA-regulated genes (reviewed in Ref. 6.Busk P.K. Pages M. Plant Mol. Biol. 1998; 37: 425-435Crossref PubMed Scopus (399) Google Scholar). Among them, those sharing a (C/T)ACGTGGC consensus sequence are found to be present in numerous ABA and/or stress-regulated genes. The elements, typified by the Em1a element (GGACACGTGGC) of wheat Em gene (7.Guiltinan M.J. Marcotte W.R. Quatrano R.S. Science. 1990; 250: 267-271Crossref PubMed Scopus (600) Google Scholar), contain the ACGT core sequence and can be considered a subset of a larger group of cis-elements known as "G-box" (CACGTG) (8.Giuliano G Pichersky E. Malik V.S. Timko M.P. Scolnik P.A. Cashmore A.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7089-7093Crossref PubMed Scopus (452) Google Scholar, 9.Menkens A.E. Schindler U. Cashmore A.R. Trend Biochem Sci. 1995; 20: 506-510Abstract Full Text PDF PubMed Scopus (326) Google Scholar). ABREs that do not contain the ACGT element are also known:e.g. "coupling element 3" (CE3) (ACGCGTGTCCTC) of barley HVA1 gene, "motif III" (GCCGCGTGGC) of rice rab16B, and a synthetic element, hex-3 (GGACGCGTGGC) (6.Busk P.K. Pages M. Plant Mol. Biol. 1998; 37: 425-435Crossref PubMed Scopus (399) Google Scholar). The sequences of these elements, which share a CGCGTG consensus, are similar to those of the former type of ABREs, except that the A of the ACGT element is replaced by G in the latter. However, the single base pair difference has been shown to be critical to the binding of plant bZIP proteins, i.e. they cannot bind to the CGCGTG element (10.Izawa T. Foster R. Chua N.-H. J. Mol. Biol. 1993; 230: 1131-1144Crossref PubMed Scopus (313) Google Scholar). Both the G-box-like ABREs and the CGCGTG-containing ABREs, here referred to as G/ABREs and C/ABREs, respectively, are functional not only in monocotyledonus plants but also in dicot plants. Other ABREs that do not belong to the G/or C/ABREs have also been reported: a Sph element-containing sequence (CGTGTCGTCCATGCAT) of the maizeC1 gene, the MYB and the MYC binding sites of theArabidopsis rd22 gene, and an element present in theCdeT27–45 gene of Craterostigma plantagineum. In general, a single unit of ABREs is not sufficient for ABA response, and a minimal sequence unit necessary and sufficient for ABA induction is composed of various combinations of the ABREs. A number of G/ABRE-binding proteins have been reported previously. EmBP-1 and TAF-1 have been isolated based on their interaction with the Em1a and a related element, motif I of rice rab16 genes, respectively (7.Guiltinan M.J. Marcotte W.R. Quatrano R.S. Science. 1990; 250: 267-271Crossref PubMed Scopus (600) Google Scholar, 11.Oeda K. Salinas J. Chua N.-H. EMBO J. 1991; 10: 1793-1802Crossref PubMed Scopus (164) Google Scholar). GBF3, originally identified as one of the G-box binding factors (GBFs) involved in the light regulation of a ribulose bisphosphate carboxylase gene (12.Schindler U. Menkens A.E. Beckmann H. Ecker J.R. Cashmore A.R. EMBO J. 1992; 4: 1261-1273Crossref Scopus (225) Google Scholar), has been cloned using the ABA-responsive, G-box element of an Arabidopsis Adh1 gene (13.Lu G. Paul A.-L. McCarty D.R. Ferl R.J. Plant Cell. 1996; 8: 847-857Crossref PubMed Scopus (66) Google Scholar). Recently, a family of embryo-specific bZIP proteins has been reported that can recognize both G/ABRE and C/ABRE (14.Kim S.Y. Thomas T.L. J. Plant Physiol. 1998; 152: 607-613Crossref Google Scholar, 15.Kim S.Y. Chung H.-J. Thomas T.L. Plant J. 1997; 11: 1237-1251Crossref PubMed Scopus (214) Google Scholar). Also, a whole array of other factors that can bind to the ACGT-containing sequences has been described (16.Foster R. Izawa T. Chua N.-H. FASEB J. 1994; 8: 192-200Crossref PubMed Scopus (299) Google Scholar, 17.Meshi T. Iwabuchi M. Plant Cell Physiol. 1995; 36: 1405-1420PubMed Google Scholar). Although ABRE binding factors have been known for some time and some of them are inducible by ABA (13.Lu G. Paul A.-L. McCarty D.R. Ferl R.J. Plant Cell. 1996; 8: 847-857Crossref PubMed Scopus (66) Google Scholar, 18.Kusano T. Berberich T. Harada M. Suzuki N. Sugawara K. Mol. Gen. Genet. 1995; 248: 507-517Crossref PubMed Scopus (93) Google Scholar, 19.Nakagawa H. Ohmiya K. Hattori T. Plant J. 1996; 9: 217-227Crossref PubMed Scopus (119) Google Scholar), several observations suggest that hitherto unidentified factors are involved in ABA-regulated gene expression during stress response, especially in vegetative tissues. ABA induction of rice rab16A and Arabidopsis rd29B genes requires de novo protein synthesis (19.Nakagawa H. Ohmiya K. Hattori T. Plant J. 1996; 9: 217-227Crossref PubMed Scopus (119) Google Scholar,20.Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1994; 6: 251-264Crossref PubMed Scopus (1571) Google Scholar), suggesting the involvement of ABA-inducible factors. In vivo binding of ABA-inducible factors has been demonstrated in the maize rab17 gene (21.Busk P.K. Jensen A.B. Pages M. Plant J. 1997; 11: 1285-1295Crossref PubMed Scopus (118) Google Scholar). In the case of rab16Bgene, currently unknown, C/ABRE binding factor(s) has been suggested to mediate ABA response through the motif III (22.Ono A. Izawa T. Chua N.-H. Shimamoto K. Plant Physiol. 1996; 112: 483-491Crossref PubMed Scopus (84) Google Scholar). Indeed, such ABA-inducible DNA-binding protein(s) has been identified in a tobacco leaf nuclear extract by in vitro binding study (23.Chung H.-J. Analysis of the 5′ Upstream Region of the Carrot Dc3 Gene: Bipartite Structure of the Dc3 Promoter for Embryo-specific and ABA-inducible Expression. Doctoral Dissertation. Texas A & M University, 1996Google Scholar). Furthermore, it has been well established by genetic studies that different ABA signaling pathways operate in seeds and in vegetative tissues, respectively (2.Leung J. Giraudat J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 199-222Crossref PubMed Scopus (997) Google Scholar), and tissue-specific ABRE binding activities have been demonstrated (24.Pla M. Vilardell J. Guiltinan M.J. Marcotte W.R. Niogret M.F. Quatrano R.S. Pages M. Plant Mol. Biol. 1993; 21: 259-266Crossref PubMed Scopus (116) Google Scholar). None of the source materials used in the previous protein-DNA interaction clonings, however, were ABA- or stress-treated young plant tissues, and thus, inducible factors that may be critical for the ABA-mediated stress response during vegetative growth phase may have been missed so far. We are interested in ABA-regulated gene expression during environmental stress response and set out to isolate relevant transcription factors. Here, we report a family of ABA-inducible bZIP proteins (designated as ABFs) that can bind to both G/ABREs and C/ABREs. ABFs are also inducible by various stress treatments, and each ABF exhibited unique induction pattern, suggesting that they are probably involved in different ABA-mediated stress-signaling pathways. Arabidopsis thaliana (ecotype Columbia) was grown at 22 °C on pots of soil (a 1:1 mixture of vermiculite and peat moss) irrigated with mineral nutrient solution (0.1% Hyponex) in 8 h light/16 h dark cycles. For RNA isolation, 4–5-week-old plants were subject to various treatments, flash-frozen in liquid nitrogen, and kept at −70 °C until needed. For ABA treatment, roots of plants were submerged after the removal of soil in a 100 μm ABA (Sigma, A 1012) solution for 4 h with gentle shaking. ABA solution was also sprayed intermittently during the incubation period. Salt treatment was performed in the same way, except that 250 mm NaCl solution was employed. For drought treatment, plants were withheld from water for 2 weeks before harvest and left on the bench after removing the soil for 1 h just before collection. For cold treatment, plants were placed at 4 °C for 24 h under dim light before harvest. Standard methods (25.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates/Wiley Interscience, New York1994Google Scholar, 26.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 27.$$$$$$ ref data missingGoogle Scholar) were used in manipulating DNA and yeast. DNA sequencing was performed on ABI 310 genetic analyzer according to the manufacturer's instructions. DNA sequence analysis was done with DNA Strider® and Generunr®, and BLAST algorithm (28.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71458) Google Scholar) was used for a data base search. Multiple sequence alignment and phylogenetic tree construction were performed with CLUSTAL W program (29.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56003) Google Scholar) available on the web. RNA was isolated according to Chomczynski and Mackey (30.Chomczynski P. Mackey K. Biotechniques. 1995; 19: 942-945PubMed Google Scholar) and further purified by LiCl precipitation followed by ethanol precipitation. For RNA gel blot analysis, 25 μg of total RNA was fractionated on 1.1% formaldehyde-agarose gel, transferred to nylon membrane (Hybond N+, Amersham Pharmacia Biotech) by the "downward capillary transfer" method (25.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates/Wiley Interscience, New York1994Google Scholar) and fixed using the Stratagene UV Crosslinker (Model 2400). Loading of equal amounts of RNAs was confirmed by ethidium bromide staining. Hybridization was at 42 °C in 5× SSC (1× SSC is 0.15m NaCl, 0.015 m sodium citrate), 5× Denhardt's solution (1× Denhardt's solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidene, 0.02% bovine serum albumin), 1% SDS, 100 μg/ml salmon sperm DNA, and 50% formamide for 24–30 h. Probes were prepared from the variable regions of ABFs. After hybridization, filters were washed twice in 2× SSC, 0.1% SDS at room temperature and three times in 0.2× SSC, 0.1% SDS for 10 min each at 65 °C. Exposure time was 7–8 days. RT-PCR was performed employing the Access RT-PCR System (Promega) using 0.5 μg of total RNA according to the manufacturer's instruction. Amplification after the first strand cDNA synthesis was 45, 35, 40, and 45 cycles for ABF1, 2, 3, and 4, respectively. ABF primers (sequences are available upon request) were from variable regions between the bZIP and the conserved regions. The actin primers used in the control reaction were from theArabidopsis actin-1 gene (accession number M20016). A lack of contaminating DNA in RNA samples was confirmed by using primer sets (ABF3 and actin) that flank introns. Poly(A)(+) RNA was isolated from total RNAs prepared from ABA- or salt-treated Arabidopsis seedlings using the Qiagen Oligotex resin. cDNA was synthesized from an equal mixture (6 μg total) of poly(A)(+) RNAs prepared from the two sources of total RNAs employing the Stratagene cDNA synthesis kit. cDNA was fractionated on a Sepharose CL-2B column, peak fractions containing cDNAs larger than 500 base pairs were pooled, and pooled cDNAs were ligated with pYESTrp2 (Invitrogen) predigested withEcoRI-XhoI. The ligation mixture was electroporated into Escherichia coli DH10B cells. The titer of this original library was 5.4 × 107 colony-forming units. Portion of the library (2 × 107) was plated on 15-cm plates at a density of 150,000 colony-forming units/plate. Cells were suspended in LB after overnight growth at 37 °C on plates and pooled together. Finally, plasmid DNA was prepared from the collected cells. pYC7-I and pSK1 (14.Kim S.Y. Thomas T.L. J. Plant Physiol. 1998; 152: 607-613Crossref Google Scholar, 15.Kim S.Y. Chung H.-J. Thomas T.L. Plant J. 1997; 11: 1237-1251Crossref PubMed Scopus (214) Google Scholar) were used as HIS3 andlacZ reporter plasmids, respectively. The G/ABRE reporter construct was prepared by inserting a trimer of Em1a element (GGACACGTGGCG) into the SmaI site of pYC7-I and theXbaI site of pSK1. To prepare reporter yeast, YPH 500 was transformed with the StuI-digested pYC7-I reporter construct. Resulting Ura+ colonies were transformed with the pSK1 construct and maintained on a SC-Leu-Ura medium. Screening of the library was performed as described (14.Kim S.Y. Thomas T.L. J. Plant Physiol. 1998; 152: 607-613Crossref Google Scholar) except that transformed reporter yeast was grown on Gal/Raf/CM-His-Leu-Trp plates instead of Glu/CM-His-Leu-Trp plates. Putative positive clones from the screen were streaked on fresh Gal/Raf/CM-His-Leu-Trp plates to purify colonies. After β-galactosidase assay, well-isolated single colonies were patched on Glu/CM-Leu-Trp-Ura plates to be kept as master plates. Galactose dependence of the His+/lacZ+ phenotype of the purified isolates was examined subsequently by comparing their growth pattern and β-galactosidase activity on Gal/Raf/CM-His-Leu-Trp and Glu/CM-His-Leu-Trp dropout plates. Yeast DNA was prepared from 1.5 ml of overnight cultures of the positive clones. PCR was performed with primers derived from the pYESTrp2 vector sequences flanking the inserts (pYESTrp forward and reverse primers). PCR products were digested withEcoRI, HaeIII, or AluI in order to group the cDNAs. For library plasmid rescue, yeast DNAs from representative clones were introduced into DH10B E. colicells by electroporation. Plasmid DNAs used in DNA sequencing and confirmation experiments were isolated from these E. colitransformants by the alkaline lysis method. For the confirmation experiment shown in Fig. 1, plasmid DNAs thus isolated were re-introduced into the yeast containing pSK1 or ABRE-pSK1, transformants were kept on Glu/CM-Leu-Trp plates, and their growth was tested after spotting 5 μl of overnight cultures (1/50 dilutions) on Gal/Raff/CM-His-Leu-Trp or Glu/CM-His-Leu-Trp plates containing 2.5 mm 3-aminotriazole. A PCR approach was used to isolate the missing 5′ portions of clone 11 and clone 19. A data base search revealed that clone 11 was part of the BAC clone F28A23 of the Arabidopsis chromosome IV. On the other hand, the 5′ portion of the clone 19 sequence was identical to the 3′ region of an EST clone, 176F17T7. Based on the sequence information, 5′ PCR primers (5′-GAAGCTTGATCCTCCTAGTTGTAC-3′ for clone 11 and 5′-ATTTGAACAAGGGTTTTAGG-GC-3′ for clone 19) were synthesized. 3′ primers (5′-TTACAATCACCCACAGAACCTGCC-3′ and 5′-GATTTCGTTGCCACTCTTAAG-3′, which are complementary to the 3′-most sequences of clones 11 and 19, respectively) were prepared using our sequence information. PCR was performed with Pwo polymerase (Roche Molecular Biochemicals) using the primer sets and 1 μg of our library plasmid DNA. After 30 cycles of reaction, the DNA fragments corresponding to the expected size of the full-length clones were gel-purified and cloned into the PCR-Script vector (Stratagene). Several clones from each PCR product were then sequenced in their entirety. The fidelity of the full-length sequences was confirmed by comparing their sequences with each other and with those of the original partial clones and the genomic clones deposited later by theArabidopsis Genome Initiative Project. To prepare GST-ABF fusion constructs, entire coding regions and the 3′-untranlated regions of ABF1and ABF3 were amplified by PCR using Pfu polymerase (Stratagene). After XhoI digestion followed by gel purification, the fragments were cloned into the SmaI-SalI sites of pGEX-5X-2 (Amersham Pharmacia Biotech). The constructs used in the transactivation assay were also prepared in a similar way. The coding regions were amplified by PCR. The resulting fragments were digested with XhoI, gel-purified, and cloned into pYX243. pYX243 was prepared by NcoI digestion, Klenow fill-in reaction,SalI digestion, and gel purification. Intactness of the junction sequences was confirmed by DNA sequencing. Recombinant ABF1 and ABF3 were prepared employing a GST purification module from Amersham Pharmacia Biotech according to the supplier's instruction. E. coli BL21 cells were transformed with the GST-ABF constructs by electroporation. To prepare bacterial extract, a single colony of transformed bacteria was inoculated in 2YT/Amp medium and grown overnight. The culture was diluted (1:100) into 250 ml of fresh media. Isopropyl-1-thio-β-d-galactopyranoside was added to the culture to a final concentration of 0.1 mm whenA 600 reached 0.7. Cells were harvested by centrifugation after further growth (1.5 h). The bacterial pellet was resuspended in 12.5 ml of phosphate-buffered saline (0.14 mNaCl, 2.7 mm KCl, 10.1 mmNa2HPO4, 1.8 mmKH2PO4, pH 7.3) and sonicated on a Branson Sonifier 250 (4 × 40-s burst at setting 5 at 80% duty cycle). The lysate was cleared of cell debris by centrifugation, and the supernatant was loaded onto a column packed with 0.125 ml (bed volume) of glutathione-Sepharose 4B resin. Wash and elution was performed as suggested by the supplier. Protein concentration was determined using the Bio-Rad protein assay kit. Production of GST-ABF1 fusion protein was confirmed by Western blotting using GST antibody. Mobility shift assay was performed as described (15.Kim S.Y. Chung H.-J. Thomas T.L. Plant J. 1997; 11: 1237-1251Crossref PubMed Scopus (214) Google Scholar). To prepare probes, oligonucleotide sets shown in Fig. 4 were annealed by boiling 100 pmol each of complementary oligonucleotides for 5 min and slowly cooling to room temperature. Portions of the annealed oligonucleotides (4 pmol of each set) were labeled by Klenow fill-in reaction in the presence of [32P]dATP. Binding reactions were on ice for 30 min, and electrophoresis was performed at 4 °C. A pool of 58-base oligonucleotides, R58, containing 18 bases of random sequence was synthesized: CAGTTGAGCGGATCCTGTCG(N)18GAGGCGAATTCAGTGCAACT, where N is a nucleotide. The random sequence is flanked byBamHI and EcoRI sites for the convenience of cloning after selection. R58 was made double strand by annealing a primer, RANR (AGTTGCACTGAATTCGCCTC) and then by extending it using Klenow fragment. For the first round of selection, 5 pmol of the double strand R58 (P0 probe) was mixed with 5 μg of the recombinant ABF1 in 100 μl of binding buffer (10% glycerol, 25 mm HEPES, pH 7.6, 100 mm NaCl, 0.5 mm EDTA, 1 mmdithiothreitol) containing 4 μg of poly(dI-dC) and incubated on ice for 30 min. The mixture was loaded onto 0.1 ml of glutathione-Sepharose 4B resin packed on a disposable column, washed with 10 volumes of the binding buffer, and eluted with 0.3 ml of 10 mmglutathione. Bound DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Amplification of the selected DNA was performed by PCR using 20 pmol each of RANF (CAGTTGAGCGGATCCTGTCG) and RANR primers in a buffer (10 mm Tris, pH 9.0, 50 mm KCl, 0.1% Triton X-100, 2.5 mm MgCl2) containing 150 μmdNTP-dATP, 4 μm dATP, 10 μCi of [32P]dATP. The reaction was carried out 20 cycles (10 s, 94 °C for 10 s, 50 °C for1 min, 72 °C). Amplified DNA was purified on a polyacrylamide gel, the band was excised after autoradiography, and DNA was eluted by the standard method to be used as a probe DNA for the next round of selection. The selection cycle was repeated two more times. For the fourth and the fifth rounds of selection, bound DNA was isolated after electrophoretic mobility shift assay (EMSA) by eluting DNA from the dried gel fragment containing the shifted bands. The amplified DNA (P5 probe) from the last selection was cloned into pBluescript (Stratagene) after EcoRI andBamHI digestion, and plasmid DNAs from 50 random colonies were sequenced. Reporter yeast containing thelacZ reporter gene (pYC7-I) with or without a trimer of the Em1a element, GGACACGTGGCG, was transformed with various pYX243/ABF constructs, and transformants were kept on Glu/CM-Leu-Ura plates. For the assay, 5 colonies from each transformant group were grown in a Glu/CM-Leu-Ura medium overnight to A 600 of approximately 1. The cultures were diluted 4–6 times with fresh media, grown further for 3 h, and pelleted by brief centrifugation. The cells were washed twice with Gal/Raf/CM-Leu-Ura medium, resuspended in 4 ml of the same medium, and grown for 4 h to induce the expression of ABFs. A 600 was measured at the end of the growth period, and 0.5 ml aliquots of the culture, in duplicates, were pelleted. The pellets were resuspended in 0.665 ml of H buffer (100 mm HEPES, 150 mm NaCl, 2 mm MgCl2, 1% bovine serum albumin, pH 7.0) and permeabilized by vortexing for 1 min after the addition of 0.055 ml each of CHCl3 and 0.1% SDS. The reaction was started by adding 0.125 ml of 40 mm stock solution of chlorophenylred-β-d-galactopyranoside, and incubation was continued at 30 °C until the color changed to red. Reactions were stopped by the addition of 0.4 ml of 1 mNa2CO3. The mixtures were microcentrifuged for 5 min to remove cell debris, and A 574 was measured. β-Galactosidase activity was expressed in Miller units. We employed a modified yeast one-hybrid system (14.Kim S.Y. Thomas T.L. J. Plant Physiol. 1998; 152: 607-613Crossref Google Scholar, 15.Kim S.Y. Chung H.-J. Thomas T.L. Plant J. 1997; 11: 1237-1251Crossref PubMed Scopus (214) Google Scholar) to isolate ABRE binding factor(s) using the prototypical ABRE, Em1a element (GGACACGTGGCG). A cDNA expression library representing 2 × 107colony-forming units was constructed in a yeast expression vector pYESTrp2 using a mixture of equal amounts of mRNAs isolated from ABA- and salt-treated Arabidopsis plants. The vector contains the B42 activation domain (31.Ma J. Ptashne M. Cell. 1987; 51: 113-119Abstract Full Text PDF PubMed Scopus (498) Google Scholar) whose expression is under the control of yeast GAL1 promoter. Thus, expression of cDNAs, which are inserted as a fusion to the activation domain, is inducible by galactose and repressed by glucose. The library DNA was used to transform a reporter yeast that harbors the ABRE-containing HIS3 andlacZ reporters. From a screen of 4 million yeast transformants, ∼40 His+ blue colonies were obtained, among which 19 isolates were characterized further. Analysis of the cDNA inserts of the positive clones indicated that they could be divided into four different groups according to their restriction patterns. Representative clones with longer inserts from each group were analyzed in more detail. First, binding of the cDNA clones to the G/ABRE in yeast was confirmed. The G/ABRE-HIS3 reporter yeast was retransformed with the library plasmid DNAs isolated from the representative clones. The growth pattern of the transformants on media lacking histidine was then examined to measure the HIS3 reporter activity. The result in Fig.1 showed that transformants obtained with all four clones could grow on a galactose medium lacking histidine but not on a glucose medium. In the same assay, the transformed yeast containing a control reporter construct lacking the ABRE could not grow on the same galactose medium. Thus, the clones could activate the HIS3 reporter gene reproducibly, indicating that they bind to the ABRE in yeast. Next, nucleotide and deduced amino acid sequences of the representative clones were determined. Clone 1, which represents two isolates, contained a cDNA insert of 1578 base pairs including a poly(A)(+) tail (GenBank™ accession number AF093544). An open reading frame (ORF) that is in-frame with the B42 domain was present within the sequence. The ORF, referred to as ABF1, contains an ATG initiation codon near the B42-cDNA junction, suggesting that it is a full-length clone. The amino acid sequence starting from the initiation codon is shown in Fig. 2. The insert of clone 2, which represents 8 isolates, is 1654 base pairs long (GenBank™ accession number AF093545), and the longest ORF including an initiation codon near the B42-cDNA junction encodes a protein of 416 amino acids (ABF2, Fig. 2). The insert of clone 11, representing 6 isolates, encoded a protein containing 434 amino acids. An ORF containing 366 amino acids was found in clone 19 cDNA. The clones were partial, however, and the missing 5′ portions were isolated using the available partial sequence information on data bases (see "Experimental Procedures"). Sequencing of the full-length clones (GenBank™ accession numberAF093546 and AF093547) showed that the original clone 11 was missing the first 20 amino acids, and thus, full-length clone 11 encodes a protein containing 454 amino acids (ABF3, Fig. 2). The longest ORF of clone 19 is composed of 431 amino acids (ABF4, Fig. 2). Analysis of the deduced amino acid sequence of ABF1 revealed that it has a basic region near its C terminus (Fig. 2). The region immediately downstream of it contains four heptad repeats of leucine, indicating that ABF1 is a bZIP protein (32.Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2547) Google Scholar). Similarly, other ABFs also have a basic region followed by a leucine repeat region (Fig. 2). The basic regions of ABF1 and ABF3 are identical to each other, and those of ABF 2 and ABF4 are also identical. The two shared basic regions are same except that one of the lysine residues of ABF1 and ABF3 is replaced by arginine in ABF 2 and ABF4 (Fig. 2). The analysis shows that a family of bZIP proteins with conserved basic regions interacts with the G/ABRE. ABFs also share several highly conserved regions ou
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