Coding-sequence evolution does not explain divergence in petal anthocyanin pigmentation between Mimulus luteus var. luteus and M. l. variegatus

Abstract

Phenotypic transitions in related taxa often share a common genetic basis, which suggests that there are constraints that shape the process of evolution at the genetic level. For example, noncoding changes in a gene may be favored relative to coding changes. Non-coding changes, which can alter context-specific gene expression, tend to have fewer pleiotropic consequences than coding changes, which affect the function of a gene product in all contexts. In this study, we evaluate the importance of coding-sequence changes to the recent evolution of a novel anthocyanin pigmentation trait in the monkeyflower genus Mimulus. The magenta-flowered Mimulus luteus var. variegatus recently gained petal lobe anthocyanin pigmentation via a single-locus Mendelian difference from its sister taxon, the yellow-flowered M. l. luteus. Mapping and functional tests previously showed that transcription factor MYB5a/NEGAN is the single gene responsible for this difference. We overexpressed the genomically encoded protein-coding sequences of MYB5a, from both M. l. luteus and M. l. variegatus, in Nicotiana tabacum leaves, in order to test their efficacy as anthocyanin-pigment activators. Quantitative image analysis of transfected tobacco leaves revealed robust anthocyanin production driven by both types of transgenes, compared to a negative control, and overall functional equivalency between the luteus and variegatus alleles. This finding supports the hypothesis that petal pigment was not gained by protein-coding changes in M. l. variegatus, but instead via non-coding cis-regulatory evolution. While constructing the transgenes needed for this experiment, we unexpectedly discovered two sites in MYB5a that appear to be post-transcriptionally edited – a phenomenon that has been rarely reported, and even less often explored, for nuclear-encoded plant mRNAs.