T and Z, partial seed coat patterning genes in common bean, provide insight into the structure and protein interactions of a plant MBW complex.

ABSTRACT

Flavonoids are secondary metabolites associated with plant seed coat and flower color. These compounds provide health benefits to humans as anti-inflammatory and antioxidant compounds. The expression of the late biosynthetic genes in the flavonoid pathway is controlled by a ternary MBW protein complex consisting of interfacing MYB, beta-helix-loop-helix (bHLH), and WD40 Repeat (WDR) proteins. P, the master regulator gene of the flavonoid expression in common bean (Phaseolus vulgaris L.), was recently determined to encode a bHLH protein. The T and Z genes control the distribution of color in bean seeds and flowers and have historically been considered regulators of the flavonoid gene expression. T and Z candidates were identified using reverse genetics based on genetic mapping, phylogenetic analysis, and mutant analysis. Domain and AlphaFold2 structure analyses determined that T encodes a seven-bladed ß-propeller WDR protein, while Z encodes a R2R3 MYB protein. Deletions and SNPs in T and Z mutants, respectively, altered the 3D structure of these proteins. Modeling of the Z MYB/P bHLH/T WDR MBW complex identified interfacing sequence domains and motifs in all three genes that are conserved in dicots. One Z MYB motif is a possible beta-molecular recognition feature (ß-MoRF) that only appears in a structured state when Z MYB is modeled as a component of a MBW complex. Complexes containing mutant T and Z proteins changed the interaction of members of the complex in ways that would alter their role in regulating the expression of genes in the flavonoid pathway.