ABFs, a Family of ABA-responsive Element Binding Factors
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 (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 M. J. M.J. Marcotte W.R. M.F. Quatrano R.S. Pages M. Plant Mol. Biol. 1993; PubMed Scopus Google Scholar). of the in the interaction young plant tissues, and inducible factors that may be critical for the ABA-mediated stress response during vegetative growth phase may have been so far. are in ABA-regulated gene expression during environmental stress response and to transcription factors. Here, we report a family of ABA-inducible bZIP proteins as that can bind to both G/ABREs and ABFs are also inducible by various stress treatments, and ABF induction suggesting that they are involved in different ABA-mediated Arabidopsis on of of and with in plants to various treatments, in and ABA of plants the of in a ABA A for with ABA also during the performed in the except that plants from for and on the the for plants for under light R. K. in Scholar, J. T. A Scholar, in and yeast. performed on genetic to the sequence analysis with and and J. Mol. Biol. 1990; PubMed Scopus Google for a base sequence and performed with 1994; PubMed Scopus Google on the isolated to and K. 1995; Google and by by of on to by the R. 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Plant Physiol. 1998; 152: 607-613Crossref Google except that reporter yeast on of from the on to single on to be as of the of the by their growth and on and from of of the PCR performed with from the sequences the and reverse PCR in to group the yeast from E. by in and isolated from these E. by the the shown in thus isolated the yeast on and their growth of on A PCR to the 5′ of and A base that of the of the Arabidopsis the other the 5′ of the sequence to the of an on the sequence 5′ PCR for and for and which are to the sequences of and using sequence PCR performed with polymerase using the and of of the to the of the and cloned the from PCR in their The of the sequences by their sequences with other and with those of the and the by theArabidopsis and the of by PCR using polymerase by the cloned the sites of The in the assay also in a similar The by The with and cloned by and of the sequences by and a from to the E. with the by a single of in and The of to the to a of by growth The in of and on a The of by and the a with of and performed as suggested by the using the protein assay of protein by using shift assay performed as described S.Y. Chung H.-J. Thomas T.L. Plant J. 1997; 11: 1237-1251Crossref PubMed Scopus (214) Google Scholar). shown in by of for and to of the of by reaction in the of Binding on for and performed A of of sequence is a The sequence is and sites for the of by a and by it using the of of the with of the in of binding of and on for The of on a with of the binding and with of by by of the performed by PCR using of and in a of The reaction for on a the and by the to be as a for the of The selection the and the of isolated electrophoretic mobility shift assay by from the the The from the selection cloned and from yeast reporter gene with a of the Em1a element, with various and on the from group in a to A of The with for and by The with in of the and for to the expression of A the of the growth and of the in The in of and by for the of of and The reaction by of of and the to by the of of The for to and A in a 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 to ABRE binding factor(s) using the prototypical ABRE, Em1a element A expression in a yeast expression using a of of isolated from and Arabidopsis plants. The the J. M. Cell. Full Text PDF PubMed Scopus Google expression is under the of yeast Thus, expression of which are as a to the is inducible by and by The to a reporter yeast that the ABRE-containing a of yeast which Analysis of the of the that they be different to their with from group in binding of the to the G/ABRE in yeast The reporter yeast with the isolated from the The growth of the on to the reporter The in showed that with grow on a but not on a In the the yeast a reporter the ABRE not grow on the Thus, the activate the reporter gene that they bind to the ABRE in yeast. and acid sequences of the which a of base a number open reading frame that is with the present the The referred to as an the suggesting that it is a The acid sequence from the is shown in The of which is base number and the an the a protein of The of a protein found in The and the 5′ isolated using the sequence on of the and showed that the the and a protein The of is composed of Analysis of the acid sequence of that it has a basic its The of it of that is a bZIP protein Science. 1988; PubMed Scopus Google Scholar). other ABFs also have a basic by a leucine The basic of and are to and those of ABF and are also The basic are except that one of the of and is replaced by in ABF and The analysis that a family of bZIP proteins with basic with the ABFs also share several the basic shown in the are in the they contain one potential The for one protein site Sci. 1990; Full Text PDF PubMed Scopus Google by a site One protein sites are also present in other The of ABFs are and in found in in binding of we performed using and a a with both proteins, and the assay of is shown in A major with a The of of to the reaction the whereas the of a and Thus, and binding to the G/ABRE in ABFs are similar to the Dc3 promoter binding factors (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 in their basic are known to not only with a G/ABRE but also with C/ABREs, we ABFs can with with the element reported except the embryo-specific E. Chua N.-H. J. Biol. 1991; Full Text PDF PubMed Google Scholar), the C/ABRE core sequence as a in an shown in a The by the of of the to the reaction and The not with a and that the binding to the Thus, and bind to a C/ABRE as in assay that and can with both C/ABREs, assay showed that they have higher to the ABF binding sites we performed a binding site selection assay R. R. 1990; PubMed Scopus Google using the on a mobility shift assay of selection the binding of to the from the of selection the cloned and The sequences are in The sequences be and to their consensus of the group I sequences except one contain an ACGT element, whereas the group sequences contain the C/ABRE The sequences of are those sharing a strong (6.Busk P.K. Pages M. Plant Mol. Biol. 1998; 37: 425-435Crossref PubMed Scopus (399) Google The group element is similar to the prototypical ABRE, Em1a whereas the group consensus is the as the G/ABREs present in ABA-inducible genes such as maize and Adh1 genes (reviewed in Ref. T.L. Chung H.-J. in Google Scholar). In some of the group sequences and the the ACGT core is replaced by consensus The group sequences share a consensus its in one the ACGT Although the element from those of group and in the the ACGT it the the case a Hence, the preferred binding sites of can be as with One of the sequences of group the C/ABRE core sequence The other group sequences also contain the C/ABRE The element in them, is by one of the group I consensus and they contain both of ABREs. sequence of group not the ACGT the of the ACGT is replaced by A. The sequence is similar to the G-box of Arabidopsis Adh1 which is for high ABA induction of the gene R. Plant Physiol. 1996; PubMed Scopus Google Scholar). Thus, with sequences the ACGT which the The selection that of the to the C/ABRE is we are in ABA-inducible factors, we ABA of ABF expression by analysis the with isolated from whereas a with from plants. with other ABF and with the from distinct with the from plants. Thus, expression of ABFs is Although are induced by the time of expression of ABFs not to other a ABA the to and to the and expression to be induced a of ABA their The induction of similar to those of and except that it the also the of various environmental stresses on the expression of The showed that expression induced by but not by other stress the other and not induced by but by high expression induced by treatments, the induction Expression of ABFs inducible also by various environmental and their induction patterns are suggesting that they in different so demonstrated that and other can bind to various ABREs and that their expression is both and Thus, ABFs have the potential to activate a large number of ABA/stress-responsive genes they have ABFs can activate an ABRE-containing reporter of ABFs cloned a yeast expression and the a yeast that a G/ABRE of the Em1a reporter gene the reporter the higher that with the with the a reporter the ABRE Thus, can transactivate the reporter gene, and the is the reporter higher the that the also can transactivate the reporter gene and transactivate the reporter gene The of higher that of the whereas showed The of assay that ABFs can activate an ABRE-containing gene in yeast. both genetic and that ABA mediates stress response in vegetative tissues, not stress responses are ABA-dependent (2.Leung J. Giraudat J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 199-222Crossref PubMed Scopus (997) Google Scholar, 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, J. Bartels D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1777) Google Scholar). to the response is the ABA regulation of gene expression through G/ABREs factors mediating and responses have been reported M.F. Science. 1998; PubMed Scopus Google Scholar, M. H. S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: PubMed Scopus Google Scholar). However, those ABA-dependent stress response the the C/ABREs have to be Among the ABRE binding factors TAF-1 is known not to be involved in ABA-responsive gene expression K. Salinas J. Chua N.-H. EMBO J. 1991; 10: 1793-1802Crossref PubMed Scopus (164) Google Scholar), whereas EmBP-1 and are embryo-specific S.Y. Chung H.-J. Thomas T.L. Plant J. 1997; 11: 1237-1251Crossref PubMed Scopus (214) Google Scholar, K. M. K. Plant Mol. Biol. 1997; PubMed Scopus Google Scholar). and a cloned inducible by are from from (13.Lu G. Paul A.-L. McCarty D.R. Ferl R.J. Plant Cell. 1996; 8: 847-857Crossref PubMed Scopus (66) Google Scholar, 19.Nakagawa H. Ohmiya K. Hattori T. Plant J. 1996; 9: 217-227Crossref PubMed Scopus (119) Google Scholar). with the of their role in ABA stress response, it is that factors may mediate ABA-responsive gene expression in vegetative tissues. In a for such transcription factors, we isolated a family of G/ABRE-binding proteins from young Arabidopsis plants with ABA high The factors, referred to as are bZIP transcription factors with basic with known factors they do not to other factors, they are similar to the (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). have been isolated from a based on their interaction with gene promoter both and The family are in their basic and their DNA-binding are similar in that they can with both of ABREs. of the sites the of ABFs are also in However, ABFs from the the basic and their in the of a they a subfamily distinct from and also from other known factors, as shown in Furthermore, their expression patterns are different from those of expression is of ABFs that related of ABRE binding factors are present in seed and in vegetative tissues, The of distinct factors in the tissues that have similar ABRE binding has been demonstrated in maize M. J. M.J. Marcotte W.R. M.F. Quatrano R.S. Pages M. Plant Mol. Biol. 1993; PubMed Scopus Google Scholar). ABFs contain them from the basic Thus, ABFs to share some other binding The do not have except that of them can and their to be may be involved in nuclear interaction with other proteins. their may the of potential sites the that it is by in vitro binding assay showed that the preferred binding site of in vitro can be as The element identified as EmBP-1 site (7.Guiltinan M.J. Marcotte W.R. Quatrano R.S. Science. 1990; 250: 267-271Crossref PubMed Scopus (600) Google is and ABA in vivo (6.Busk P.K. Pages M. Plant Mol. Biol. 1998; 37: 425-435Crossref PubMed Scopus (399) Google Scholar). with the that is and has that can activate a large number of ABA/stress-responsive genes Also, can bind to other ABREs the C/ABREs, the of potential genes. The to C/ABREs, cannot be that factors other ABFs with C/ABREs with higher The expression of ABFs that they are involved in the regulation of ABA-responsive genes expression requires protein on the of de novo protein ABA-responsive gene can be one protein synthesis and the other of protein synthesis (reviewed in Ref. K. Yamaguchi-Shinozaki K. Curr. Opin. Biotechnol. 1996; 7: 161-167Crossref PubMed Scopus (397) Google Scholar). Expression of Arabidopsis rd29B and genes, for requires protein whereas ABA induction of the wheat Em gene ABA of ABF thus that ABFs are to the expression of the former of genes. ABF may in different ABA-dependent Although are ABA-inducible and can bind to ABREs, they are by various environmental stresses expression is induced by ABF and by high salt, and by high and The of the be that is involved in whereas and in stress on the other to in stress In ABFs in their ABA induction Expression of induced and the of its whereas induction of other ABFs and their a The of ABA-dependent pathways has been demonstrated in Arabidopsis by genetic analysis (2.Leung J. Giraudat J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 199-222Crossref PubMed Scopus (997) Google M. Plant Cell. 1997; 9: PubMed Scopus Google Scholar). that transcription factors are to in these through ABREs. ABA-dependent gene expression is critical to plant growth and productivity. Here, we reported a family of transcription factors that with elements mediating Although their in to be here suggest that they are to be involved in various ABA-mediated stress can bind to ABREs can transactivate an ABRE-containing reporter expression is induced by ABA and by various environmental and for their critical reading of the also for during of