A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History.

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the "tip of the iceberg" since thousands of sequences remain uncharacterized in this enzyme family alone.

How petals change their spots: cis-regulatory re-wiring in Clarkia (Onagraceae).

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 F2 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.

Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae).

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 showed that the dihydroflavonol-4-reductase 2 (Dfr2) gene has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicated the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development as a result of early expression of Dfr2 and the flavonoid 3' hydroxylase 1 (F3'h1) gene. Pigments in the background appear later, as a result of later expression of Dfr1 and the flavonoid 3'-5' hydroxylase 1 (F3'5'h1) genes. 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.

Positive selection for single amino acid change promotes substrate discrimination of a plant volatile-producing enzyme.

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.