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

Biologists have long been interested in understanding genetic constraints on the evolution of development. For example, noncoding changes in a gene might be favored relative to coding changes due to being less constrained by pleiotropic effects. Here 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. Previous work showed that the differentially expressed transcription factor gene MYB5a/NEGAN is the single causal gene. However, it was not clear whether MYB5a coding-sequence evolution (in addition to the observed patterns of differential expression) might also have contributed to increased anthocyanin production in M. l. variegatus. Quantitative image analysis of tobacco leaves, transfected with MYB5a coding sequence from each taxon, revealed robust anthocyanin production driven by both alleles. Counter to expectations, significantly higher anthocyanin production was driven by the allele from the low-anthocyanin M. l. luteus. Together with previously-published expression studies, this supports the hypothesis that petal pigment in M. l. variegatus was not gained by protein-coding changes, but instead solely via non-coding cis-regulatory evolution. Finally, 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.

Methods
A 1-mL disposable syringe with the needle removed was used to deliver A. tumefaciens cells containing overexpression transgenes to the leaves of young (1–3 months) N. tabacum. The top of the leaf was held firmly while the leaf underside was injected until liquid had visibly spread past the site of injection (100–200μL). The M. l. luteus and M. l. variegatus alleles of transcription factor gene MYB5a were infiltrated in pairs, alternating which transgene was injected into the left versus the right side of each leaf. pGFPGUSPlus negative controls were evenly distributed between the two sides of other leaves.

Leaves were imaged 3–12 days after infiltration in a dark room with a Nikon D3500 DSLR camera with an 18–55mm lens, illuminated by a Sylvania Ceramic Metal Halide bulb. VGG Image Annotator was used on .jpeg images to demarcate the infiltration boundaries and injection site.

A custom Python program imported RAW image files for processing, using a modified version of the MacDuff color chart detection algorithm (https://github.com/mathandy/python-macduff-colorchecker-detector) to automatically detect a reference color chart. Image pixel values were converted into normalized reflectance values based on a linear fit of the red, green, and blue signal strengths to the color chart.