Evidence for Direct Activation of an Anthocyanin Promoter by the Maize C1 Protein and Comparison of DNA Binding by Related Myb Domain Proteins

Abstract

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.

The basic helix–loop–helix domain of maize R links transcriptional regulation and histone modifications by recruitment of an EMSY-related factor

“…4). Such a model had been predicted from extensive transient expression experiments and mutational analyses of the A1 promoter (9,11,19,28), but a direct in vivo tethering of C1 to the A1 promoter was never shown before. Most significant from a mechanistic perspective, however, our results support a model in which C1 is primarily responsible for specifying the promoters to which R needs to be recruited, while R furnishes a docking platform for the recruitment of additional factors to the complex, including RIF1, as shown in this study.…”

“…They participate in the transcriptional regulation of the anthocyanin pathway genes through the cooperation with the R2R3-MYB transcription factor C1 or its paralog, PL1 (7). C1 and R/B physically interact through the MYB domain of C1 and the N-terminal region of R (which does not contain the bHLH motif) (8,9), and C1 makes direct DNA contacts with specific cis-regulatory elements, which, in the case of the A1 gene (encoding dihydroflavonol reductase, DFR), correspond to the high-and low-affinity P1 binding sites ( ha PBS and la PBS, respectively) (10,11). R/B belong to the group IIIf of plant bHLH factors (12), a subfamily that is shared with similar anthocyanin regulators in various plants as well as with the Arabidopsis GL3/EGL3 regulators of epidermal cell patterning (13).…”

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

“…First, AN2 can induce expression of an1 but not of jaf13 (Figure 7). Second, activity of the P1 protein can be enhanced by AN1 but not by JAF13 ( Figure 5B) or R1 (Grotewold, 1995;Sainz et al, 1997). Third, using two-hybrid screens in yeast, we identified a protein that interacts with the bHLH domain of AN1 but fails to recognize JAF13 in yeast (A.…”

“…In maize suspension cells, forced expression of P1 alone is sufficient to induce transcription of a1 (Grotewold et al, 1994;Bruce et al, 2000), and this induction cannot be enhanced by coexpression of R1; this indicates that P1, unlike C1, functions independent of R1-like bHLH factors (Grotewold, 1995;Sainz et al, 1997). Transcripts of an1, an2, jaf13, dfrA, and gapdh were detected by quantitative RT-PCR in the corolla and tube, anthers, ovaries, and sepals from flowers at various developmental stages and from leaves, stems, roots, and pistils of the wild-type line V30, as indicated above the lanes.…”

“…In two-hybrid assays in yeast and maize cells, B1 interacts with C1, which suggests that both proteins are in the same transcription complex (Goff et al, 1992). In vitro, C1 binds with low affinity to functional cis -acting elements in the promoters of structural genes (Sainz et al, 1997), but thus far, no DNA binding activity has been detected for R1. Whether this is because R1 needs to dimerize with an as yet unknown partner or because the C1/R1 complex interacts in vivo with the promoter of an unknown "intermediate" regulator is unclear at this stage.…”