Functional specialization of stomatal bHLHs through modification of DNA-binding and phosphoregulation potential

Transcription factor duplication events and subsequent specialization can drive evolution by facilitating biological innovation and developmental complexity. Identification of sequences that confer distinct biochemical function in vivo is an important step in understanding how related factors could refine specific developmental processes over time. Functional analysis of the basic helix-loop-helix (bHLH) protein SPEECHLESS, one of three closely related transcription factors required for stomatal lineage progression in Arabidopsis thaliana, allowed a dissection of motifs associated with specific developmental outputs. Phosphorylated residues, shown previously to quantitatively affect activity, also allow a qualitative shift in function between division and cell fate-promoting activities. Our data also provide surprising evidence that, despite deep sequence conservation in DNA-binding domains, the functional requirement for these domains has diverged, with the three stomatal bHLHs exhibiting absolute, partial, or no requirements for DNA-binding residues for their in vivo activities. Using these data, we build a plausible model describing how the current unique and overlapping roles of these proteins might have evolved from a single ancestral protein.