MYB5a/NEGAN activates petal anthocyanin pigmentation and shapes the MBW regulatory network in Mimulus luteusvar. variegatus

Abstract

Much of the visual diversity of angiosperms is due to the frequent evolution of novel pigmentation patterns in flowers. The gene network responsible for anthocyanin pigmentation, in particular, has become a model for investigating how genetic changes give rise to phenotypic innovation. In the monkeyflower genus Mimulus, an evolutionarily recent gain of petal lobe anthocyanin pigmentation in M. luteus var. variegatus was previously mapped to genomic region pla2. Here, we use DNA sequence analysis and spatiotemporal patterns of gene expression to identify MYB5a - homologous to the NEGAN transcriptional activator from M. lewisii - as a likely candidate gene within the pla2 region. Transgenic manipulation of gene expression confirms that MYB5a is both necessary and sufficient for petal lobe anthocyanin pigmentation. The deployment of MYB5a/NEGAN to the petal lobe stands in contrast to its more restricted role as a nectar guide anthocyanin activator in other Mimulus species. Transcriptome sequencing of a MYB5a RNAi line reveals the degree to which other regulators of the anthocyanin pathway - including R3 MYB repressors and bHLH and WD40 co-activators - are responsive to the level of expression of MYB5a. Overall, this work reveals that a genetically simple change, which we hypothesize to be a regulatory mutation in cis to MYB5a, has cascading effects on gene expression, not only on the genes downstream of MYB5a but also on all of its known partners in the anthocyanin regulatory network. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/030536v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@120d538org.highwire.dtl.DTLVardef@8e6085org.highwire.dtl.DTLVardef@162f684org.highwire.dtl.DTLVardef@26860f_HPS_FORMAT_FIGEXP M_FIG Graphical abstract.Solid black arrows indicate the direction (though not magnitude) of gene expression change, following RNAi knockdown of MYB5a/NEGAN in M. l. variegatus. The number of black arrows corresponds to the number of gene copies identified in the transcriptome. Grey symbols denote positive and negative regulatory interactions. RTO is an R3 MYB protein that inhibits anthocyanin biosynthesis by sequestering bHLH proteins away from the MBW complex. C_FIG