Barley BLZ2, a Seed-specific bZIP Protein That Interacts with BLZ1 in Vivo and Activates Transcription from the GCN4-like motif of B-hordein Promoters in Barley Endosperm

Abstract

A barley endosperm cDNA, encoding a DNA-binding protein of the bZIP class of transcription factors, BLZ2, has been characterized. The Blz2 mRNA expression is restricted to the endosperm, where it precedes that of the hordein genes. BLZ2, expressed in bacteria, binds specifically to the GCN4-like motif (GLM; 5′-GTGAGTCAT-3′) in a 43-base pair oligonucleotide derived from the promoter region of a Hor-2 gene (B1-hordein). This oligonucleotide also includes the prolamin box (PB; 5′-TGTAAAG-3′). Binding by BLZ2 is prevented when the GLM is mutated to 5′-GTGctTCtc-3′ but not when mutations affect the PB. The BLZ2 protein is a potent transcriptional activator in a yeast two-hybrid system where it dimerizes with BLZ1, a barley bZIP protein encoded by the ubiquitously expressed Blz1 gene. Transient expression experiments in co-bombarded developing barley endosperms demonstrate that BLZ2 transactivates transcription from the GLM of the Hor-2 gene promoter and that this activation is also partially dependent on the presence of an intact PB. A drastic decrease in GUS activity is observed in co-bombarded barley endosperms when using as effectors equimolar mixtures of Blz2 and Blz1 in antisense constructs. These results strongly implicate the endosperm-specific BLZ2 protein from barley, either as a homodimer or as a heterodimer with BLZ1, as an important transcriptional activator of seed storage protein genes containing the GLM in their promoters. A barley endosperm cDNA, encoding a DNA-binding protein of the bZIP class of transcription factors, BLZ2, has been characterized. The Blz2 mRNA expression is restricted to the endosperm, where it precedes that of the hordein genes. BLZ2, expressed in bacteria, binds specifically to the GCN4-like motif (GLM; 5′-GTGAGTCAT-3′) in a 43-base pair oligonucleotide derived from the promoter region of a Hor-2 gene (B1-hordein). This oligonucleotide also includes the prolamin box (PB; 5′-TGTAAAG-3′). Binding by BLZ2 is prevented when the GLM is mutated to 5′-GTGctTCtc-3′ but not when mutations affect the PB. The BLZ2 protein is a potent transcriptional activator in a yeast two-hybrid system where it dimerizes with BLZ1, a barley bZIP protein encoded by the ubiquitously expressed Blz1 gene. Transient expression experiments in co-bombarded developing barley endosperms demonstrate that BLZ2 transactivates transcription from the GLM of the Hor-2 gene promoter and that this activation is also partially dependent on the presence of an intact PB. A drastic decrease in GUS activity is observed in co-bombarded barley endosperms when using as effectors equimolar mixtures of Blz2 and Blz1 in antisense constructs. These results strongly implicate the endosperm-specific BLZ2 protein from barley, either as a homodimer or as a heterodimer with BLZ1, as an important transcriptional activator of seed storage protein genes containing the GLM in their promoters. cultivar alcohol dehydrogenase I days after pollination −90-bp minimal 35S CaMV promoter endosperm box electrophoretic mobility shift assay Gal4 responsive elements in a Gal1 truncated promoter Gal4 activation domain Gal4 DNA binding domain GCN4-like motif β-glucuronidase gene imidazole glycerol phosphate dehydratase gene β-galactosidase gene prolamin box cauliflower mosaic virus 35S promoter 3′-terminator sequence of the nopaline synthase gene base pair(s) nucleotide(s) polymerase chain reaction Hordeins, the major storage proteins of barley seeds, are prolamins specifically synthesized in the starchy endosperm and are classified according to their mobility in SDS-electrophoretic gels into three major classes: B, C, and D, with the B fraction representing ∼75% of the total hordein content in most barley cultivars (cv.)1. All the hordeins are structurally related, and their genes presumably derive from a common ancestor by gene duplication and subsequent divergent evolution (1Kreis M. Shewry P.R. Shewry P.R. Barley: Genetics, Molecular Biology and Bio/Technology. CAB International, Wallingford, U.K.1992Google Scholar). The coordinate expression of all hordein genes suggests common regulatory mechanisms of transcription that should involve bothcis-acting motifs in their promoters andtrans-acting transcription factors (2Shewry P.R. Napier J.A. Tatham A.S. Plant Cell. 1995; 7: 945-956Crossref PubMed Scopus (740) Google Scholar). A conserved cis-acting motif that is found in most storage protein gene promoters of seeds is the endosperm box (EB; Refs. 3Sørensen M.B. Cameron-Mills V. Brandt A. Mol. Gen. Genet. 1989; 217: 195-201Crossref Scopus (71) Google Scholar and4Bevan M. Colot V. Hammond-Kosack M. Holdworth M. Torres M. Smith C. Grierson C. Beggs K. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1993; 342: 209-215Crossref PubMed Scopus (15) Google Scholar), a bipartite motif located around 300 bp upstream of the translation initiation ATG codon, that contains two distinct nuclear protein binding sites: the prolamin box (PB, 5′-TGTAAAG-3′), also called the endosperm motif (EM), and a GCN4-like motif (GLM, 5′-(G/A)TGA(G/C)TCA(T/C)-3′), which resembles the binding site of the yeast transcription factor GCN4 (5Hill D.E. Hope I.A. Macke J.P. Struhl K. Science. 1986; 234: 451-457Crossref PubMed Scopus (211) Google Scholar, 6Müller M. Muth J.R. Gallusci P. Knudsen S. Maddaloni M. Motto M. Schmitz D. Sørensen M.B. Salamini F. Wettstein D. Thompson R.D. J. Plant Physiol. 1995; 145: 606-613Crossref Scopus (25) Google Scholar). These two motifs are present in B- and C-hordein promoters, whereas only the PB is present in that of D-hordeins. Functional analysis of a native C-hordein promoter by particle bombardment of developing barley endosperms (7Müller M. Knudsen S. Plant J. 1993; 6: 343-355Crossref Scopus (165) Google Scholar) have demonstrated that the GLM is the dominant cis-acting element and that the PB exerts a silencing effect. However, both GLM and PB from the promoter of a Hor-2 gene (B1-hordein) are essential positive elements conferring a high level of transcriptional activity to the minimal 35S CaMV promoter (Δ35S) in bombarded developing endosperms from barley (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar). Much of what is known about the genetic and molecular mechanisms regulating cereal seed storage genes comes from work on maize, where a bZIP protein, OPAQUE 2 (O2; Refs. 9Hartings H. Maddaloni M. Lazzaroni N. Di Fonzo N. Motto M. Salamini F. Thompson R. EMBO J. 1989; 8: 2795-2801Crossref PubMed Scopus (182) Google Scholar and 10Schmidt R.J. Burr F.A. Aukerman M.J. Burr B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 46-50Crossref PubMed Scopus (346) Google Scholar), has been shown to bind to and activate transcription from an ACGT core motif adjacent to the PB in the promoter of the 22-kDa class of zein genes (11Lohmer S. Maddaloni M. Motto M. Di Fonzo N. Hartings H. Salamini F. Thompson R.D. EMBO J. 1991; 10: 617-624Crossref PubMed Scopus (189) Google Scholar, 12Schmidt R.J. Ketudat M. Aukerman M.J. Hoschek G. Plant Cell. 1992; 4: 689-700Crossref PubMed Scopus (288) Google Scholar). In wheat and barley, current knowledge about the transcription factors involved in the regulation of storage protein genes is not as complete as in maize although bZIP proteins, such as SPA from wheat and BLZ1 from barley, have been described in endosperm that are able to interact with the GLM binding site in prolamin genes. Whereas SPA is seed-specific, BLZ1 is also expressed in other tissues and both have been shown to transiently transactivate appropriate reporter genes in planta (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar, 13Albani D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar). Recently, the O2 protein has been shown to interact in vitro with another maize endosperm-specific factor (PBF) of the Dof (DNA-binding with onefinger) class that recognizes the PB motif in the −300 region of the 22-kDa zein promoter (14Vicente-Carbajosa J. Moose S.P. Parsons R.L. Schmidt R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7685-7690Crossref PubMed Scopus (305) Google Scholar). Its barley orthologue (BPBF) transactivates transcription from the PB element of a nativeHor-2 promoter in co-bombarded barley developing endosperm (15Mena M. Vicente-Carbajosa J. Schmidt R.J. Carbonero P. Plant J. 1998; 16: 53-62Crossref PubMed Google Scholar). With the emerging picture of the EB as a refinedcis-regulatory element whereby several nuclear proteins interact, it is crucial the identification of the factors that can participate in this complex and coordinate gene regulation in seeds. BZIP factors are particularly interesting because these proteins bind DNA motifs via dimer formation, either as homo- or heterodimers besides interacting with other regulatory proteins (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar, 16Foster R. Izawa T. Chua N.-H. J. 8: PubMed Scopus Google Scholar, N. Cell. 1990; PubMed Scopus Google Scholar, Aukerman M.J. Schmidt R.J. Plant Cell. 1993; PubMed Scopus Google Scholar, T. 1993; 7: PubMed Scopus (288) Google Scholar). bZIP factor can bind to the The regulatory of a bZIP factor is the of a in a promoter and with other regulatory factors and other expressed in a the the and of a barley encoding a endosperm-specific bZIP transcriptional BLZ2 that a high of sequence to SPA from wheat D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar). The BLZ2 protein in binds in vitro to the GLM in the EB of a promoter and transiently transactivates transcription from the GLM in the the barley developing transcriptional BLZ2 also an intact PB in the of the GLM In BLZ2 transcription in the yeast two where it can heterodimers with in the and in the endosperms and and and in and endosperms particle bombardment experiments from and A from barley endosperms (15Mena M. Vicente-Carbajosa J. Schmidt R.J. Carbonero P. Plant J. 1998; 16: 53-62Crossref PubMed Google Scholar) representing after of the The by J. T. Molecular A Scholar) and using as a a from the barley Blz1 J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar) most of bZIP This DNA by the 2 in and the The in and in and the in of the system the according to and in the DNA using the system and the DNA sequence of DNA and protein with the from barley tissues as described M. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Google Scholar). using the of the J. T. Molecular A Scholar). A shown in as a Blz2 The with a a a A. J. M. 7: Scholar) and with a barley 8: PubMed Scopus Google Scholar) as a the barley BLZ2 protein in the S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a transcriptional of the sequence in the site of the a Blz2 shown in by the polymerase chain reaction using the as a that and containing the translation initiation of the of the Blz2 and a that the of the in This into an after the expression into of BLZ2 expression and of protein as described (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar). the with as The containing the EB from the Hor-2 promoter and two mutated of the GLM or the PB described in the by that These with by the reaction DNA and from an experiments as described (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar), using of The and which the alcohol dehydrogenase I promoter to the binding domain or to DNA activation domain to or Blz1 or with derived from The of and glycerol phosphate reporter genes the of a truncated Gal1 promoter that elements BLZ2 and BLZ1 able to the bZIP domain of Blz2 to shown in that and by a using as oligonucleotide site and as site into of the The into S. containing the of the Blz1 the the region the region encoding the bZIP domain and that of the BLZ2 a transcriptional activator in the Blz2 shown in with the and the and into the and of The region of Blz2 shown in by of the and and into and of All by and by the 1992; 13: Scholar) and β-galactosidase by the assay R. Smith J.A. Struhl K. in Molecular Scholar) and in a The reporter a containing the β-glucuronidase reporter gene Plant Mol. Biol. Scopus Google Scholar) the of a promoter and to the 3′-terminator of the nopaline synthase gene In the and upstream of this the of the sequence of the endosperm box from promoter containing the element mutated in the GLM and containing the element mutated in the PB The promoter as a The by in the or antisense the of the 35S CaMV promoter to the of the maize gene and by the V. D. Bio/Technology. 1991; 9: Scopus Google the cDNA, by with the and the with and and and into the the antisense the and and into a restricted with the The and antisense Blz1 as described (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar). bombardment with a according to J.R. Plant 1993; Scopus Google Scholar). in as described by and Plant 1991; 10: PubMed Scopus Google Scholar) by of with 2 of of and of In all or of the reporter and the appropriate of the the on 2 with and in of of and of a of from the developing barley endosperms on containing of and bombardment with of the endosperms according to Plant Mol. Biol. Scopus Google Scholar). a and the GUS activity in assay expressed as the of The with the GUS activity of protein with a of not The presence in wheat and maize of endosperm-specific bZIP proteins, SPA and O2 H. Maddaloni M. Lazzaroni N. Di Fonzo N. Motto M. Salamini F. Thompson R. EMBO J. 1989; 8: 2795-2801Crossref PubMed Scopus (182) Google Scholar, 10Schmidt R.J. Burr F.A. Aukerman M.J. Burr B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 46-50Crossref PubMed Scopus (346) Google Scholar, 13Albani D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar), that activate transcription with the GLM or ACGT of the EB in storage protein gene promoters, to their barley such a the bZIP region of the ubiquitously expressed barley Blz1 (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar) as a to a (15Mena M. Vicente-Carbajosa J. Schmidt R.J. Carbonero P. Plant J. 1998; 16: 53-62Crossref PubMed Google Scholar) from developing endosperm positive after and of containing the The and the DNA sequence of the in this in A Blz2 is a gene not and a bZIP protein that contains and a molecular of are found upstream of the ATG This by and other transcription factor have a in translation regulation (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar, S. Maddaloni M. Motto M. Salamini F. Thompson R.D. Plant Cell. 1993; Google Scholar). The BLZ2 protein has a bZIP domain with a region by A motif involved in T. M. Cell. 1992; PubMed Scopus Google Scholar), two in that involved in transcription and a nuclear that the complete region of the bZIP domain Schmidt R.J. Plant Cell. 1992; 4: PubMed Scopus Google Scholar) are also The BLZ2 protein is the of wheat SPA D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar) because the protein, and in their bZIP BLZ2 is also to the barley BLZ1 protein the in the bZIP to O2 from maize, and to from and to from maize and has but with from and from and of The on the of the proteins and the sequence that in that these proteins a of of the BLZ2 protein and other bZIP transcription using the using the in a of the BLZ2 protein and other bZIP transcription using the The and of expression of the Blz2 gene by from endosperms from to and from and days after with a the mRNA only in the endosperm BLZ2 a of hordein gene the of Blz2 expression to and the of the hordein this the with a the sequence of a Hor-2 gene. The present and In the and expressed in endosperm The of Blz2 and hordein are with the that Blz2 an activator of the hordein genes. the of BLZ2 in the regulation of hordein gene BLZ2 of binding specifically to the GLM the EB sequence from the promoter of gene in This DNA because it a conserved sequence in the −300 promoter region of genes that are expressed in the endosperm M. Holdsworth M.J. Bevan M.W. EMBO J. 1993; Scopus Google Scholar). of the BLZ2 protein, with the and to bind to this by shown in two when BLZ2 with the the motifs this that by BLZ2, containing mutations in the GLM or in the PB in the BLZ2 able to bind to the the when the GLM mutated to as when using the oligonucleotide However, an intact not the in vitro binding because mutations in this not affect the complex of all by experiments and of oligonucleotide In these and whereas to the BLZ2 binding to or A and These results that BLZ2 binds specifically to the GLM sequence the EB of hordein promoters. The of the observed in vitro BLZ2 and the GLM in by the of BLZ2 on expression in co-bombarded barley shown in GUS reporter containing in their promoters EB either in the GLM or in the endosperms transiently by particle bombardment with these or in with as a in B, of and in an of about in the GUS activity with that by the The by a GUS expression that of the by the by the barley endosperm factors that bind to the bipartite EB The promoter the in the GLM of the element a GUS activity the and is not by the co-bombarded The mutated in the a GUS expression of of that by the both with or the BLZ2 These results demonstrate that BLZ2 transcription the GLM in barley endosperm, and that this activation is also partially dependent on the presence of an intact PB in the The and Blz1 in the barley endosperm to BLZ2 and BLZ1 heterodimers in this by the yeast two-hybrid using as a the bZIP domain of BLZ2, to the and as a the BLZ1 or of it to the these two proteins expressed in yeast interact, the complex a transcription which is of binding to the in the promoters of reporter and their The are in A. The encoding the bZIP domain of Blz2 to the in the and the to the in the into the yeast shown in B all with containing the encoding of both proteins expression of and reporter that BLZ2 bZIP domain with BLZ1 in activation of the when yeast with the two and B, or when only with the or with the B, and a positive the Blz1 in the B, that been shown to an activator in the yeast system (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar). The in by not activate promoter not BLZ2 also in yeast as a transcriptional the or in the and it into S. shown in and BLZ2 is a transcriptional and the region is to activate transcription of the reporter genes. that BLZ2 with BLZ1 in yeast and that BLZ2, as BLZ1 (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google Scholar), transcriptional activation in barley endosperm from the GLM of hordein promoters, to the of BLZ2 and BLZ1 to such activation in developing barley this experiments of using the as reporter and the Blz2 as both in the and antisense shown in BLZ2 and BLZ1, to the GUS able to transactivate the GUS expression from the and in the level of activation observed when endosperms co-bombarded with either of the two effectors or with the equimolar of Blz2 in the or with Blz1 antisense the GUS activity with the reporter In of endosperms with the equimolar of both Blz2 and Blz1 antisense a that in the complete of GUS the The GUS observed when the effectors in the and the drastic decrease when using in antisense not an because GUS to the promoters of the barley synthase encoding genes and and J. Carbonero P. Plant Physiol. 1997; PubMed Scopus Google Scholar), as as to the not either or (8Vicente-Carbajosa J. Oñate L. Lara P. Dı́az I. Carbonero P. Plant J. 1998; 13: 629-640Crossref PubMed Scopus (75) Google expression analysis in barley activity expressed as of endosperm and as GUS expression the of several promoters in co-bombarded barley endosperms using as the in or in antisense developing endosperms in three experiments co-bombarded with of the reporter and a to the in or in antisense as GUS activity expressed as of endosperm and as in a Transient GUS expression the of several promoters in co-bombarded barley endosperms using as the in or in antisense developing endosperms in three experiments co-bombarded with of the reporter and a to the in or in antisense as have a from barley that an endosperm-specific bZIP transcription factor that transcription in the barley with the GLM sequence from prolamin gene promoters. In BLZ2 can as an activator in yeast where it is of with The of expression of Blz2 with a in the transcription regulation of endosperm-specific genes mRNA expression with that of encoding which the major fraction of storage proteins in barley seeds, this expression The EB of the promoter of a Hor-2 gene is of PB and that are conserved in the promoters of prolamin genes of the such are encoding B- and in barley A. J. M. 7: Scholar, A. A. Cameron-Mills V. Scopus Google Scholar, J. Knudsen S. M. Cameron-Mills V. Plant Mol. Biol. 1991; PubMed Scopus Google Scholar), and in wheat V. Bevan M.W. Thompson R.D. EMBO J. 6: PubMed Google Scholar, M. J.A. T. M. D. 13: Scholar) and in M. Shewry P.R. Plant Mol. Biol. 1991; PubMed Scopus Google Scholar). results that BLZ2 binds in vitro in a to the GLM the EB of a promoter and that mutations the sequence of the GLM the the of BLZ2 in expression in the barley endosperm where it is is known that in the or expression by gene promoters are V. Bevan M.W. 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Plant J. 6: PubMed Scopus Google Scholar, T. R. Chua N.-H. J. Mol. Biol. 1993; PubMed Scopus Google Scholar). This with the the transcriptional planta of BLZ2 and BLZ1, strongly the that both proteins are not the of the that transcription activation the However, the of other factors to the endosperm box In this with the barley factor (15Mena M. Vicente-Carbajosa J. Schmidt R.J. Carbonero P. Plant J. 1998; 16: 53-62Crossref PubMed Google Scholar) major in their of as with homo- or heterodimer bZIP are this BLZ2 and wheat SPA a sequence that is not restricted to the bZIP endosperm-specific expression and DNA binding which that these genes results and the about the of the GLM in the regulation of storage protein genes and a of the endosperm-specific of bZIP proteins as transcriptional in cereal seeds of the J. and I. are of the in DNA and J. and L. with the of the