And R-Dependent Expression of the Maize Bzi Gene Requires Sequences with Homology to Mammalian Myb and Myc Binding Sites
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The Plant Cell, Vol. 3, 317-325, March 1991 O 1991 American Society of Plant Physiologists CI- and R-Dependent Expression of the Maize BzI Gene Requires Sequences with Homology to Mammalian myb and myc Binding Sites Bradley A. Roth,’ Stephen A. Goff,* Theodore M. Klein,3 and Michael E. Fromm4 Plant Gene Expression Center, United States Department of Agriculture/University of California, Berkeley, 800 Buchanan Street, Albany, California 9471 O Tissue-specific expression of the maize anthocyanin Bronze-7 (627) gene is controlled by the products of several regulatory genes. These include C7 or PI and R or B that share homology to the myb proto-oncogenes and myc-like genes, respectively. 621 expression in embryo tissues is dependent on C7 and an R-sc allele of R. Transient expression from mutated and deleted versions of the 627 promoter fused to a luciferase reporter gene was measured in C7, Rscm2 embryos after gene transfer by microprojectiles. This analysis revealed that the sequences between -76 base pairs (bp) and -45 bp and a 9-bp AT-rich block between -88 bp and -80 bp were critical for 627 expression. The -76 bp to -45 bp region includes two short sequences that are homologous to the consensus binding sites of the myb- and myc-like proteins. Site-specific mutations of these “myb” and “myc” sequences reduced k7expression to 10% and 1% of normal, respectively. Additionally, a trimer of a 38-bp oligonucleotide containing these myb and myc sites increased the expression of a cauliflower mosaic virus 35s minimal promoter by 26-fold. This enhancement was dependent on both C1 and R. Because the sites critical for 627 expression are homologous to the myb and myc consensus binding sequences and the C1 and R proteins share homology with the myb and myc products, respectively, we propose that C7 and R interact with the 627 promoter at these sites. INTRODUCTION The extensive genetics of the maize anthocyanin biosyn- The predicted PI-encoded and C7-encoded proteins are thetic pathway and the isolation of many of its structural homologous to each other (K. Cone, unpublished results) and regulatory genes have provided an ideal system to and to proteins of the animal myb proto-oncogene family study the regulation of gene expression in plants. The (Paz-Ares et al., 1987). The myb protein has been shown enzymes of the pathway are encoded by a set of genes, to bind DNA specifically (Biedenkapp et al., 1988; Howe including A7, Bronze-7 (Bzl), and C2. These structural et al., 1990; Oehler et al., 1990). Also, myb proteins contain genes are coordinately regulated by several regulatory loci, acidic regions that have been found to function as tran- which condition the tissue-specific production of anthocy- scriptional activators (Klempnauer et al., 1989; Weston anin pigment (Coe et al., 1988). In general, anthocyanin and Bishop, 1989). Similarly, the C7 carboxyl terminus pigment formation requires the expression of at least two contains an acidic region (Paz-Ares et al., 1987) that has classes of regulatory genes: a PI or C7 gene and a member been shown to function to activate transcription in plants from the R or B gene families (Coe et al., 1988). Light can when fused to the yeast Ga14 DNA binding domain (Goff partially substitute for the requirement for P1 (expressed et al., 1991). primarily in the plant body) and in some instances for C7 The R and B gene families both show extensive diversity (expressed in the aleurone and embryo). among alleles, each of which directs a different tissue- specific pattern of pigment formation (Coe et al., 1988). The R and B genes have been shown to encode very ’ To whom correspondence should be addressed. Current Ad- similar proteins (Dellaporta et al., 1988; Chandler et al., dress: Pioneer Hi-Bred International, Inc., P.O. Box 38, Johnston, 1989; Ludwig et al., 1989; Perrot and Cone, 1989) and IA 50131. * Current Address: Department of Biology, lnstitute of Molecular contain regions that are homologous to myc-like proteins Biology, University of Oregon, Eugene, OR 97403. (Ludwig et al., 1989). The myc-related, helix-loop-helix Current Address: E.I. DuPont, Agricultura1 Products, Experimen- proteins have been shown to bind specifically to DNA and tal Station, 402/4250, Wilmington, DE 19880. to function as transcriptional activators (Murre et al., Current Address: Monsanto Company, Plant Sciences, 700 1989a; Beckmann et al., 1990; Davis et al., 1990; Henthorn Chesterfield Village Parkway, St. Louis, MO 63198. et al., 1990). 318 The Plant Cell The BZl structural gene encodes UDPg1ucose:flavonol than in nonpermissive tissues (c7, r; Cl,r; c7, R aleurones 3-0-gIucosyltransferase, which catalyzes one of the last or embryos). Therefore, if the sites in the Bzl promoter steps in the synthesis of anthocyanin pigment (Dooner and that respond to C7 and R are removed by deletion or Nelson, 1977). Both C7 and R were shown to be necessary mutagenesis, a similar large drop in relative activity would for the production of the Bz7 enzyme and mRNA in the be expected in permissive tissues. Because deletion of aleurone of the kernel (Dooner and Nelson, 1979; Cone et essential core promoter elements might also cause similar al., 1986). In embryos, both C7 and certain alleles of R, decreases in promoter activity, further experiments would such as Rscm2, are required for Bzl expression (Dooner be required to distinguish between these possibilities. and Nelson, 1977,1979). Bzl mRNA also has been shown to be regulated by 6 and PI in the husks of mature plants (Gerats et al., 1984; Chandler et al., 1989; Sullivan et al., Luciferase Expression from Bzí 5’-Deleted Promoters 1989). Recently, we showed that the C7,R genotype of maize aleurone and embryo tissues regulated the expres- To locate the critical regions for Bz7 promoter activity, a sion of a Bzl promoter that was attached to a firefly series of 5‘ deletions of the Bzl promoter were con- luciferase reporter gene after DNA transfer by micropro- structed. Each chimeric gene included a Bzl 5’-deleted jectiles (Klein et al., 1989). Luciferase expression from the promoter (with end points from -407 bp to +2 bp), the Bzl promoter in Cl,R aleurone tissue was 100-foldhigher Bzl first intron, the firefly luciferase coding region, and the than the luciferase expression in aleurones mutant for c7 Bzl 3‘ region, as shown in Figure 1A. Fifteen Bzl 5‘- or r. Luciferase expression in C7, Rscm2 embryos was deleted promoters were introduced into bz7, C7 , Rscm2 100-fold higher than in C7,R embryos or those mutant for embryos, which are permissive for the expression of c7. or r. pBzl L. Additionally, pBzl L and pLBz3’, a promoterless In the present study, we utilize DNA transfer by micro- derivative of pBzl L (Klein et al., 1989), were introduced projectiles into intact C7,Rscm2 embryos to measure the as positive and negative controls, respectively. In all bom- levels of expression from various mutated and deleted Bzl bardments, the plasmid pBzl CAT (see Methods) was promoters. Our results indicate that at least two short coprecipitated onto the microprojectiles with each experi- regions upstream of the TATA box are necessary for the mental plasmid. The chloramphenicol acetyltransferase expression of Bz7. One of these regions contains sites (CAT) enzyme activity directed by this plasmid was used similar to the consensus sites for DNA binding by the myb- as a control to verify delivery of DNA and to standardize like and myc-like proteins (Sen and Baltimore, 1986; Bie- the luciferase activity. denkapp et al., 1988), which share homology with the Cl Figure IAshows the average luciferase-to-CAT ratio for and R products, respectively. Fusions of the Bzl promoter each 5’-deleted promoter construct, as well as the relative upstream of an Adh7 core promoter indicate that these activity compared with pBzl L. Relative activities from regions are necessary for an 18-fold increase in expression 102% to 34% were observed in the 12 constructs with of the Adhl core promoter. Additionally, a trimer of an deletion end points between -407 bp and -1 14 bp. The oligonucleotide containing the Bz7 “myb” and “myc” sites relatively small decreases in expression with these con- fused to a minimal promoter increased gene expression structs indicate that Cl and/or R most likely do not interact 26-fold when cobombarded into embryogenic maize callus with these regions of the Bz7 promoter. The relative activ- (c7,r) with B-peru and C7 expressed from the cauliflower ities observed for the -80 bp, -60 bp, and +2 bp deleted mosaic virus (CaMV) 35s promoter. This enhancement of promoter constructs were all 2% or less. These results the minimal promoter expression is dependent on C7 and suggest that the important cis-acting regions required for B (homologous to R). Thus, these experiments define the expression of the Bz7 promoter are located within 114 bp cis-acting sites in the BZl promoter that respond to regu- of the mRNA start site. lation by Cl and R. Analysis of Expression of Bzí Promoters Containing Interna1 Deletions and Mutations RESULTS A set of Bz7 3’-deleted promoters was constructed (see Experimental Rationale Methods) to help define in more detail the important re- gions of the Bzl promoter. These 3’-deleted promoters A chimeric Bz7 promoter-luciferase plasmid, pBzl L, was were combined with the 527 5’-deleted promoter series shown previously to be regulated in a manner similar to to create a set of promoters with internal deletions (Figure that of the endogenous Bz7 gene after transfer by micro- 1B). Each of these internal deletion constructs was copre- projectiles to intact aleurone and embryo tissues (Klein et cipitated with pBzl CAT onto microprojectiles and bom- al., 1989).