A novel R3 MYB transcriptional repressor associated with the loss of floral pigmentation in Iochroma

Abstract

Losses of floral pigmentation represent one of the most common evolutionary transitions in flower color, yet the genetic basis for these changes has been elucidated in only a handful of cases. Here we used crossing studies, bulk-segregant RNA sequencing, phylogenetic analyses and functional tests to identify the gene(s) responsible for the transition to white flowers in Iochroma loxense. Crosses between I. loxense and its blue-flowered sister species, I. cyaneum, suggested that a single locus controls the flower color difference and that the white allele causes a nearly complete loss of pigmentation. Examining sequence variation across phenotypic pools from the crosses, we found that alleles at a novel R3 MYB transcription factor were tightly associated with flower color variation. This gene, which we term MYBL1, falls into a class of MYB transcriptional repressors and, accordingly, higher expression of this gene is associated with downregulation of multiple anthocyanin pigment pathway genes. We confirmed the repressive function of MYBL1 through stable transformation of Nicotiana. The mechanism underlying the evolution of white flowers in I. loxense differs from that uncovered in previous studies, pointing to multiple mechanisms for achieving fixed transitions in flower color intensity.

Developmental control of convergent floral pigmentation across evolutionary timescales

“…In this species, the white morphs are nearly 100‐fold down‐regulated for Dfr and Ans relative to the purple‐flowered individuals (Figure ) and sit nearest the white species in expression space (Figure ). The coordinated downregulation of these two genes is consistent with a mutation in their shared MYB transcriptional regulators . Although the expression differences in the remaining taxa appear too minor to drive a loss of pigmentation alone, it is important to note that white morphs might arise through the combined action of multiple segregating regulatory or coding mutations.…”

“…Our flavonoid profiling confirms that the white morphs that appear periodically in populations of normally pigmented species share the same biochemical signature as white species, that is, they lack the colorful anthocyanins while still producing the upstream flavonols (Figures ). This biochemical change is tightly associated with downregulation of downstream genes (DFR, ANS) in the white species but not in the white morphs (Figures and ). Instead, the overall expression patterns for white morphs are most similar to their pigmented counterparts (Figures and ).…”

“…By contrast, fixed species‐level losses of floral anthocyanins across multiple clades have uniformly been attributed to mutations in MYB transcription factors, which coordinately down‐regulate the multiple pathway genes . For instance, the shift from purple to white flowers in the I loxense lineage (pictured in Figure ) was caused by a dominant gain‐of‐function mutation in an R3 MYB repressor, resulting in losses of DFR and ANS expression . This bias toward fixation of mutations in MYB transcription factors has been attributed to their high specificity (in terms of DNA targets, tissues and timing), which presumably results in lower potential for deleterious pleiotropy …”

“…Anthocyanins are responsible for red, blue, and purple colors in most flowering plants, and mutations that disrupt their production result in white to yellow flowers, depending on the background levels of yellow carotenoids and flavonoid copigments . Fixed losses of floral anthocyanin pigmentation are perhaps among the best studied phenotypic transitions in plants, and they appear to consistently involve mutations in the MYB transcription factors that regulate the downstream steps of the pathway, for example, DFR and ANS (Figure ) . While there are fewer analyses of segregating “acyanic” (white or yellow) morphs within pigmented species, studies‐to‐date point to a wider range of causal changes including both trans ‐regulatory mutations and loss‐of‐function mutations in enzyme‐coding genes …”

“…7,18,19 Fixed losses of floral anthocyanin pigmentation are perhaps among the best studied phenotypic transitions in plants, and they appear to consistently involve mutations in the MYB transcription factors that regulate the downstream steps of the pathway, for example, DFR and ANS ( Figure 1). 11,21 While there are fewer analyses of segregating "acyanic" (white or yellow) morphs within pigmented species, studies-to-date point to a wider range of causal changes including both trans-regulatory mutations and loss-of-function mutations in enzyme-coding genes. [22][23][24] Building on existing knowledge of the flavonoid pathway ( Figure 1) and its role in flower color, we examine pathway gene expression and floral pigmentation in the Andean clade, Iochrominae (Solanaceae).…”