Summary Petal spots are widespread in Angiosperms and are often implicated in pollinator attraction. Clarkia gracilis petals each have a single red-purple spot that contrasts against a pink background. The position and presence of spots in C. gracilis are determined by the epistatic interaction of alleles at two, as-yet-unidentified loci.We used HPLC to identify the different pigments produced in the petals, and qualitative and quantitative RT-PCR to assay for spatio-temporal patterns of expression of different anthocyanin pathway genes.We found that spots contain different pigments from the remainder of the petal, being composed of cyanidin/peonidin-based, instead of malvidin-based anthocyanins. Expression assays of anthocyanin pathway genes show that Dfr2 has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicate the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development due to early expression of Dfr2 and F3′h1. Pigments in the background appear later, due to later expression of Dfr1 and F3′5′h1.The evolution of this spot production mechanism appears to have been facilitated by duplication of the Dfr gene and to have required substantial reworking of the anthocyanin pathway regulatory network.

We used a combined evolutionary and experimental approach to better understand enzyme functional divergence within the SABATH gene family of methyltransferases (MTs). These enzymes catalyze the formation of a variety of secondary metabolites in plants, many of which are volatiles that contribute to floral scent and plant defense such as methyl salicylate and methyl jasmonate. A phylogenetic analysis of functionally characterized members of this family showed that salicylic acid methyltransferase (SAMT) forms a monophyletic lineage of sequences found in several flowering plants. Most members of this lineage preferentially methylate salicylic acid (SA) as compared with the structurally similar substrate benzoic acid (BA). To investigate if positive selection promoted functional divergence of this lineage of enzymes, we performed a branch-sites test. This test showed statistically significant support (P<0.05) for positive selection in this lineage of MTs (dN/dS=10.8). A high posterior probability (pp=0.99) identified an active site methionine as the only site under positive selection in this lineage. To investigate the potential catalytic effect of this positively selected codon, site-directed mutagenesis was used to replace Met with the alternative amino acid (His) in a Datura wrightii floral-expressed SAMT sequence. Heterologous expression of wild-type and mutant D. wrightii SAMT in Escherichia coli showed that both enzymes could convert SA to methyl salicylate and BA to methyl benzoate. However, competitive feeding with equimolar amounts of SA and BA showed that the presence of Met in the active site of wild-type SAMT resulted in a >10-fold higher amount of methyl salicylate produced relative to methyl benzoate. The Met156His-mutant exhibited little differential preference for the 2 substrates because nearly equal amounts of methyl salicylate and methyl benzoate were produced. Evolution of the ability to discriminate between the 2 substrates by SAMT may be advantageous for efficient production of methyl salicylate, which is important for pollinator attraction as well as pathogen and herbivore defense. Because BA is a likely precursor for the biosynthesis of SA, SAMT might increase methyl salicylate levels directly by preferential methylation and indirectly by leaving more BA to be converted into SA.

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A long-standing question in evolutionary developmental biology is how new traits evolve. Although most floral pigmentation studies have focused on how pigment intensity and composition diversify, few, if any, have explored how a pattern element can shift position. In the present study, we examine the genetic changes underlying shifts in the position of petal spots in Clarkia. Comparative transcriptome analyses were used to identify potential candidate genes responsible for spot formation. Co-segregation analyses in F individuals segregating for different spot positions, quantitative PCR, and pyrosequencing, were used to confirm the role of the candidate gene in determining spot position. Transient expression assays were used to identify the expression domain of different alleles. An R2R3Myb transcription factor (CgMyb1) activated spot formation, and different alleles of CgMyb1 were expressed in different domains, leading to spot formation in different petal locations. Reporter assays revealed that promoters from different alleles determine different locations of expression. The evolutionary shift in spot position is due to one or more cis-regulatory changes in the promoter of CgMyb1, indicating that shifts in pattern element position can be caused by changes in a single gene, and that cis-regulatory rewiring can be used to alter the relative position of an existing character.