It is well established that a network of three classes of proteins consisting of R2R3MYB, bHLH factors and WD40 repeat protein acted in concert as a ternary complex (i.g. MBW protein complex) to activate the flavonoid-based pigment biosynthetic pathway in most high plants. Several additional functions evolved in rosids: e.g. trichome patterning, root hair patterning and seed coat mucilage production in Arabidopsis (A. thaliana) or Arabis (A. alpina) and seed hair formation in cotton (G. hirsutum). New roles of MBW complexes controlling epidermal cell fate in rosids may have diverged since the evolutionary separation of rosid and asterid, although the details of this are still not clear.
Previous studies in our lab revealed novel stereochemistry of MBW components, i.g. alternative dimers TTG1-GL3 and GL1-GL3, which revised the conventional TTG1-GL3-GL1 ternary model. However, it raises one major question: what are the evolutionary implications of such alternative dimers formation among MBW components in plants? In this study, we characterized the stereochemistry of MBW proteins in different plant species by triple LUMIER assay. Using the inter-relation of MBW components as the criterion, we achieved a highly accordant phylogenetic tree suggesting the evolutionary relevance of this novel stereochemistry of MBW components. Potential critical sites in bHLH proteins accounting for diversed MBW stereochemistry were predicted.
In Arabidopsis, MBW genes which control trichome and root hair patterning traits are assumed to evolve from the duplication and diversification of flavonoid controlling genes, therefore trichome and root hair traits are considered as evolutionary current inventions. However, the exact evolving order of these traits still remains to be confirmed. To better define functional divergence of the MBW proteins in the five TTG1 related traits, we performed cross-species complementary assays with MBW homologs in Arabidopsis mutants.
Among MBW protein complexes in Arabidopsis, AtTTG1-AtGL3-AtGL1 is considered to be the predominant regulatory complex in leaf trichome formation. This regulatory unit is not only represented by a single trimeric complex (synergetic inter-relation) but also by two alternative dimers (antagonistic inter-relation) that in turn regulate different downstream genes. Probably even more important is the finding that different promoters become activated depending on the relative concentration of these three proteins, as this should translate into different ratios of alternative dimers and trimers. In this study, we attempted to simulate regulatory models in the context of differential proportion of alternative dimers through quantitatively determining AtTTG1 and AtGL1 competing for the binding to AtGL3. These experimental studies were complemented by mathematical modeling by Anna Deneer, Waageningen
