Ohio State University
The maize anthocyanin accumulation pathway has been well characterized and its regulation is well understood. The Myb domain proteins C1 or PL require the presence of the bHLH proteins R or B to activate transcription of the structural genes of the pathway. It is not clear however how the activity of these transcription factors is modulated. In both Petunia and Arabidopsis factors that act upstream of the known regulators of flavonoid accumulation and trichome development have been found. In the case of Petunia, a cytosolic WD-repeat protein is involved in the regulation of accumulation of anthocyanins, and mutants in An11 do not accumulate pigments in the corolla. Overexpression of An2, the Petunia orthologue of C1, is able to activate the dfr promoter in an An11 mutant background suggesting that An11 acts upstream of An2 (de Vetten et al., Genes & Dev. 11:1422-1434, 1997). TTG is another WD-repeat protein involved in the accumulation of anthocyanins as well as trichomes in Arabidopsis (Walker et al., Plant Cell 11: 1337-1349, 1999). The effects of ttg mutations are more pleiotropic than those of An11 (Koornneef, Arabidopsis Inf. Serv. 18: 45-51, 1981). Lloyd et al. showed that overexpressing the maize R gene in ttg mutants restored both anthocyanin accumulation and trichome formation, suggesting that TTG is higher in the regulatory hierarchy than the bHLH proteins (Lloyd et al., Science 266: 436-439, 1994).
To determine whether a similar hierarchy of regulator proteins is present in maize, we used degenerate primers to conserved regions of An11 to generate a PCR from maize seedling polyA+, which was RNA and then used as a probe to screen cDNA and genomic maize libraries. A gene called Mp1 with no introns and encoding a protein of 410 amino acids (45 kD) was identified. MP1 is also a WD-repeat protein and it shares high sequence identity with AN11 and TTG (Table 1). A northern blot of polyA+ mRNA from various maize tissues indicated that MP1 is expressed throughout the entire plant. This is consistent with the expression patterns of AN11 and TTG (de Vetten et al., Genes & Dev. 11:1422-1434, 1997; Walker et al., 1999). Analysis of the Mp1 sequence indicates that there is no nuclear localization signal and suggests that it might encode a cytosolic protein, similar to An11. Preliminary experiments suggest that Mp1 is incapable of complementing a ttg mutant.
Mapping experiments have positioned Mp1 to the long arm of chromosome 5 close to Pac1, a recently identified locus which is involved in anthocyanin accumulation in the aleurone. Examination of the level of expression of the structural genes and the regulators of the pathway in pac1 mutants revealed that the levels of expression of only the former but not the latter were dramatically decreased (Selinger and Chandler, Plant Cell 11: 5-14). This is a similar situation to the one reported for An11 in which the levels of An2, Jaf13 and An1 were not decreased in an11 mutants (de Vetten et al., Genes & Dev. 11:1422-1434, 1997). In addition to failure to accumulate anthocyanins neither pac1 or an11 mutants show any other phenotype which contrasts with the more pleiotropic effects of ttg mutations. Whether Mp1 and Pac1 are the same gene is an open question that needs further investigation.
Phylogenetic analysis indicates that MP1 is closely related to AN11 and TTG, and that these proteins form a family of proteins that are widespread among animals and plants as suggested by de Vetten et al. Figure 1. WD-repeat proteins are usually part of signal transduction cascades, and are involved in protein-protein interactions. It is possible that this new family of WD-repeat proteins is involved in a signal transduction cascade that ultimately modifies bHLH and/or Myb domain proteins enabling them to activate transcription and switch on the pathways in which they are involved.
Table 1. Percent identities of MP1 to other WD-repeat proteins.
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