Figure 4

The sites of positive selection in OryzaTPS1 helped to determine which sesquiterpene products were formed. Hierarchical cluster analysis was performed for ObTPS1, OrTPS1 and the 31 ObTPS1 mutants. For each enzyme, the relative abundance of each sesquiterpene product was expressed as a percentage of the total amount of the four products. The color range represented percentages from 0% (a target compound was not detected at the expected retention time) to 100% abundance. Average values from three replicates were used. On the right, simplified chromatograms were depicted showing the relative abundance of the products of ObTPS1 (representing the ancestral (E)-β-caryophyllene synthesizing activity, ECS), ObTPS1-C429G (the only single mutant producing more germacrene A than ObTPS1), ObTPS1-F318 + V433I (the typical double mutant producing detectable germacrene D) and ObTPS1-F318 + C429G + V433I (the only triple mutant producing comparable amounts of germacrene A, germacrene D and (E)-β-caryophyllene). Products were identified as 1, β-elemene; 2, (E)-β-caryophyllene; 3, β-humulene; 4, germacrene D by comparison of their retention times and mass spectra to those of authentic standards. *Unidentified sesquiterpenoids. Note: β-elemene was produced as a thermal rearrangement product from germacrene A in the GC injector (Additional files 4 and 5).