The benzoylcyclohexane-1,3-diones, the triketones, are potent bleaching herbicides whose structure-activity relationships and physical properties are substantially different from classical bleaching herbicides, which affect phytoene desaturase. The first clue to their unique mechanism of action was the discovery that rats treated with a triketone were found to be tyrosinemic. Additionally, examination of the rat urine revealed the accumulation of p-hydroxyphenylpyruvate (HPP) and p-hydroxyphenyllactate. These results suggested that this chemically induced tyrosinemia was the result of the inhibition of p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), and this suggestion was confirmed when a triketone was shown to be a potent inhibitor of rat liver HPPD. In plants, HPPD is a component of the biosynthetic pathway to plastoquinone (PQ), which in turn is a key cofactor of phytoene desaturase. The expectation that triketone-treated plants should accumulate tyrosine while having reduced PQ levels was dramatically demonstrated in the meristematic tissue of ivyleaf morningglory. Plant HPPD, like the mammalian enzyme, was inhibited in vitro by triketones. These biochemical effects provide evidence that the triketone herbicidal mechanism of action is HPPD inhibition leading to a deficiency of PQ, a key cofactor for carotenoid biosynthesis. Other chemical classes of bleaching herbicides were also examined for their ability to elevate tyrosine and deplete PQ as a definitive means of establishing their mode of action and for delineating the structural and physical chemical requirements for an HPPD herbicide. Evidence is provided to support the claim that a 2-benzoylethen-1-ol substructure is the minimum substructure required for a potent HPPD inhibitor.

With rapid progress being made in deciphering plant genomic sequences, determining the function of these genes is one of the main challenges that plant molecular biologists face today. Herbicidal inhibitors have been very useful for understanding gene function in at least two examples, represented by herbicidal inhibitors of hydroxyphenylpyruvate dioxygenase (HPPD) and deoxyxylulosephosphate reductoisomerase (DXR). In the first, an albino mutant of Arabidopsis isolated during the study of carotenoid biosynthesis was found to have an intact carotenoid biosynthetic pathway. A number of “bleaching herbicides” in development at about the same time (e.g., sulcotrione) produced similar symptoms by strongly inhibiting HPPD, a key enzyme in plastoquinone biosynthesis. Examination of the Arabidopsis mutant revealed that the HPPD gene had been inactivated in the albino plants. Inhibition of the HPPD pathway also led to reduced levels of tocopherol (vitamin E), an end product of the pathway. Further studies and manipulation of the pathway produced plants with significantly higher levels of vitamin E. This result is a clear demonstration of how an herbicidal inhibitor was able to lead to the identification of a gene that was responsible for a particular phenotype. As a second example, identification of fosmidomycin as a specific inhibitor of DXR in the recently elucidated nonmevalonate pathway of isopentenyl pyrophosphate (IPP) biosynthesis was instrumental in furthering the understanding of an important route to synthesis of many important terpenoid products.