Evolution of flux control in the glucosinolate pathway in Arabidopsis thaliana

Network characteristics of biochemical pathways are believed to influence the rate of evolutionary change in constituent enzymes. One characteristic that may affect rate heterogeneity is control of the amount of product produced by a biochemical pathway or flux control. In particular, theoretical analyses suggest that adaptive substitutions should be concentrated in the enzyme(s) that exert the greatest control over flux. Although a handful of studies have found a correlation between position in a pathway and evolutionary rate, these investigations have not examined the relationship between evolutionary rate and flux control. Given that genes with greater control will experience stronger selection and that the probability of fixation is proportional to the selective advantage, we ask the following: 1) do upstream enzymes have majority flux control, 2) do enzymes with majority flux control accumulate adaptive substitutions, and 3) are upstream enzymes under higher selective constraint? First, by perturbing the enzymes in the aliphatic glucosinolate pathway in Arabidopsis thaliana with gene insertion lines, we show that flux control is focused in the first enzyme in the pathway. Next, by analyzing several sequence signatures of selection, we also show that this enzyme is the only one in the pathway that shows convincing evidence of selection. Our results support the hypothesis that natural selection preferentially acts on enzymes with high flux control.