The maize Myb transcription factor C1 depends on the basic helix-loop-helix (bHLH) proteins R or B for regulatory function, but the closely related Myb protein P does not. We have used the similarity between the Myb domains of C1 and P to identify residues that specify the interaction between the Myb domain of C1 and the N-terminal region of R. Substitution of four predicted solvent-exposed residues in the first helix of the second Myb repeat of P with corresponding residues from C1 is sufficient to confer on P the ability to physically interact with R. However, two additional Myb domain amino acid changes are needed to make the P regulatory activity partially dependent on R in maize cells. Interestingly, when P is altered so that it interacts with R, it can activate the Bz1 promoter, normally regulated by C1 ؉ R but not by P. Together, these findings demonstrate that the change of a few amino acids within highly similar Myb domains can mediate differential interactions with a transcriptional coregulator that plays a central role in the regulatory specificity of C1, and that Myb domains play important roles in combinatorial transcriptional regulation. Combinatorial interactions between transcription factors are of central importance to regulation of gene expression in eukaryotes. These interactions can either modulate transcription factor activity or contribute to the biological specificity of factors with very similar DNA-interaction motifs. Elucidation of the mechanisms by which proteins with very similar DNA-binding domains achieve regulatory specificity remains a fundamental question in biology today.Proteins containing the Myb-homologous DNA-binding domain are widespread in eukaryotes (reviewed in refs. 1 and 2). The vertebrate c-myb gene plays an essential regulatory role in the proliferation and differentiation of hematopoietic cells. Besides c-myb, at least two other myb-related genes (A-myb and B-myb) are present in vertebrates (3). The products of these genes have Myb domains, each consisting of three head-to-tail Myb motifs (R1, R2, and R3). Oncogenic versions of c-myb, such as v-myb, contain only R2 and R3, as do hundreds of plant Myb-domain proteins (4). Myb domains formed by the R2 and R3 Myb motifs bind DNA. Each Myb motif contains three ␣-helices, and the third helix of each Myb motif makes sequencespecific DNA contacts. The second and third helices of each Myb motif form a helix-turn-helix structure when bound to DNA, similar to motifs found in the repressor and in homeo domains (5). In addition to their well-established roles in DNA binding, Myb domains are also emerging as important protein-protein interaction motifs. These Myb domain-mediated proteinprotein interactions play key roles in the biological specificity of the corresponding factors (6-13). However, the mechanisms by which protein-protein interactions contribute to the regulatory specificity of Myb domain proteins are poorly understood.In f lowering plants, several hundred genes containing the conserved Myb DNA-binding domain have b...

The enzyme-encoding genes of two classes of maize flavonoid pigments, anthocyanins and phlobaphenes, are differentially regulated by distinct transcription factors. Anthocyanin biosynthetic gene activation requires the Myb domain C1 protein and the basic helix-loop-helix B or R proteins. In the phlobaphene pathway, a subset of C1-regulated genes, including a7, are activated by the Myb domain P protein independently of BIR. We show sequence-specific binding to the a1 promoter by C1 in the absence of B. Activation is decreased by mutations in the C1 DNA binding domain or in a1 sequences bound by C1, providing direct evidence for activation of the anthocyanin biosynthetic genes by C1. The two C1 binding sites in the a7 promoter are also bound by P. One site is bound with higher affinity by P relative to C1, whereas the other site is bound with similar lower affinity by both proteins. Interestingly, either site is sufficient for C1 plus B/R or P activation in vivo, demonstrating that differences in DNA binding affinities between P and C1 are insufficient to explain the differential requirement for B. Results of DNA binding site-selection experiments suggest that C1 has a broader DNA binding specificity than does P, which may help C1 to activate a more diverse set of promoters.

SummaryA major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.