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Comparative Study
. 1998 Jun;117(2):437-45.
doi: 10.1104/pp.117.2.437.

Activation of the maize anthocyanin gene a2 is mediated by an element conserved in many anthocyanin promoters

M L Lesnick  1 , V L Chandler
Affiliations
Comparative Study

Activation of the maize anthocyanin gene a2 is mediated by an element conserved in many anthocyanin promoters

M L Lesnick et al. Plant Physiol. 1998 Jun.

Abstract

Two transcription factors, C1 (a Myb-domain protein) and B (a basic-helix-loop-helix protein), mediate transcriptional activation of the anthocyanin-biosynthetic genes of maize (Zea mays). To begin to assess the mechanism of activation, the sequences required for C1- and B-mediated induction have been determined for the a2 promoter, which encodes an anthocyanin-biosynthetic enzyme. Analysis of a series of 7- to 13-base-pair substitutions revealed two regions crucial for activation. One region, centered at -99, contained a C1-binding site that abolished C1 binding. The other crucial region was adjacent, centered at -91. C1 binding was not detected at this site, and mutation of this site did not prevent C1 binding at -99. An oligonucleotide dimer containing these two crucial elements was sufficient for C1 and B activation of a heterologous promoter. These data suggest that activation of the anthocyanin genes involves C1 and another factor binding at closely adjacent sites. Mutating a previously postulated anthocyanin consensus sequence within a2 did not significantly reduce activation by C1 and B. However, sequence comparisons of the crucial a2 regions with sequences important for C1- and B-mediated activation in two other anthocyanin promoters led to a revised consensus element shared by these promoters.

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Figures

Figure 2
Figure 2
Activity of a2 promoters with substitution mutations. A, Mutations 1 through 7 were created using PCR to substitute the specific 7- to 13-bp sequence indicated. These mutations maintain the spacing found in the wild-type a2 promoter. The sequence of each of these mutations is shown in the bar below its corresponding number. The last line represents a deletion, with the gap corresponding to the deleted bp. Each of these mutations was assayed for its ability to be activated by C1 and B in the context of the −112-bp promoter. The percentage activation of each of these promoters in transient expression assays relative to the level of activation of the wild-type −112-bp a2 promoter (set at 100%) is shown at the right. Error bars represent se; n = 12. B, Mutations 2 and 3, as well as the deletion shown, were tested in the context of the −287-bp a2 promoter. The percentage activation relative to the −287-bp a2 promoter (set at 100%) is shown at the right. se is indicated for each construct; n = 12.
Figure 6
Figure 6
a2-promoter sequences sufficient for C1 and B activation of a heterologous promoter. A synthetic promoter composed of a dimer of an oligonucleotide containing a2 sequences from −121 to −81 in front of a truncated, −59-bp CaMV 35S promoter was tested in the maize transient transformation system. The induction in the presence of C1 and B is shown. The boxed region is the C1-binding site that overlaps the crucial region identified by mutation 2. The lines represent the two regions that when mutated in the context of the −112 region had dramatic effects on C1- and B-mediated activation.
Figure 1
Figure 1
C1 and B activation of the 1.9-kb a2 promoter or 5′ deletion derivatives in tissue-cultured maize cells. A, Schematic representations of the plasmids used in the transient transformation assay. The CaMV 35S promoter controls the expression of the C1 and B proteins. The reporter plasmid consists of the a2 promoter or mutant derivatives fused to the luciferase-coding region. Details of these constructs are given in Methods. B, The 1.9-kb a2 promoter was cotransformed with either an empty vector control plasmid containing the CaMV 35S promoter without any coding region (–), or with plasmids designed to express B, C1, or a mutant derivative of C1, D101E (defective in DNA binding). Bars represent activation of the a2-luciferase reporter gene, which was obtained by dividing the luciferase activity obtained in each bombardment by the activity from the transformation control included in each bombardment (for details, see Methods). Error bars represent se; n = 12. The −287 a2 promoter, which is equivalent to the 1.9-kb a2 promoter (C), was used for the C1-D101E experiment. Activity is normalized to wild-type C1 and B activation of the same promoter. C, 5′ Deletions of the a2 promoter were generated and tested for activation by C1 and B in transient transformation assays as described in B. Histograms represent the percentage activation in the presence of C1 and B, normalized to the activation observed with the 1.9-kb a2 promoter, set at 100%. Error bars represent se; n = 12.
Figure 3
Figure 3
An ARE overlaps with regions crucial for C1 and B induction of the a2 promoter. A, The sequence of the −112 to −41 a2 promoter is shown, with results from the linker-scanner mutagenesis shown below. Bars above the sequence indicate the anthocyanin consensus sequence suggested by Tuerck and Fromm (1994) (Previous Consensus), or as proposed here (New Consensus). B, Computer alignment (Devereux et al., 1984) of three characterized anthocyanin promoters reveals common sequences among all three. The consensus site derived from this sequence similarity is shown above. WT, Wild type.
Figure 4
Figure 4
C1 binds specifically to the a2 promoter in at least three locations. A, A radiolabeled fragment of the a2 promoter, spanning −161 to +5, was mixed with purified C1 protein in the presence or absence of competitor oligonucleotides or C1 and non-C1 antibodies. The specific competitor is a C1-binding site from the a1 promoter (−76 to −47). The mutant competitor is the same fragment of a1, but with 2-bp substitutions that reduce activation (Grotewold et al., 1994) and C1 DNA binding (Sainz et al., 1997). B through D, Methylation interference experiments using purified C1 Myb protein and the indicated end-labeled oligonucleotides of the a2 promoter (see Methods). Lanes U contain methylation-interference reactions purified from unbound labeled probe. Lanes B contain reactions purified from bound probe. The sequence to the left of each gel corresponds to the sequence of the oligonucleotide, with the protected region boxed.
Figure 5
Figure 5
The binding of C1 to radiolabeled oligonucleotides containing either the wild-type (WT) sequence or mutations 2 (Mut2) or 3 (Mut3). Each labeled probe was used in three binding reactions, with probe alone and with 1 and 3 μg of C1 Myb protein, as indicated above each lane. The diagram below shows the wild-type oligonucleotide sequence, with the location of the two mutations boxed. Figure 2 contains the sequence changes in the mutant derivatives. The levels of activation of these mutations by C1 and B in the context of the −112-bp a2 promoter is shown below.
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