Hypostatin, a new small molecule inhibitor of plant cell expansion, is glyco-activated in vivo

Natural variation in humans in the response to a bioactive or drug-like small organic molecule is defined as pharmacogenetic variation. Studies have shown that variation in both drug metabolism genes and drug target loci can cause inter-individual variation in drug response. For example, sequence differences in UDP-glucuronosyl transferases (UGTs), which glycosylate xenobiotics, can affect drug sensitivity by affecting rates of drug detoxification. Although this subject has been explored extensively in humans, the biological pervasiveness of pharmacogenetic variation had not been systematically examined. If pervasive, pharmacogenetic variation in model systems could be used for both functional studies and to gain deeper insight into the mechanisms of this important form of natural variation. To examine this question, 8 geographically diverse Arabidopsis accessions were screened on a 10,000 member chemical library. I focused on new inhibitors of cell expansion in the etiolated hypocotyl. In total, 742 chemicals (7.4%) caused greater than 20% inhibition of hypocotyl cell expansion. 11 weak polymorphic chemicals were uncovered, as well as a strong polymorphic hypocotyl cell expansion inhibitor, named hypostatin. My work has firmly established the existence of pharmacogenetic variation in Arabidopsis.;To understand the mechanism of this variation, I characterized the molecular basis of variation in hypostatin sensitivity. Genetic analysis of F1, F2 and recombination inbred lines (RIL) of crosses between Col and Ler (which show differential sensitivity) showed that hypostatin resistance is determined by a single recessive locus, named HYR1. Map-based cloning showed that HYR1 encodes a UDP-glycosyltransferase (UGT71B2) that has no substrate documented and a known pharmacogenetic factor related humans. My research has shown that hypostatin-sensitive Arabidopsis accessions encode functional HYR1 proteins that glucosylate hypostatin in vivo and convert it into an activated form. Hypostatin resistant accessions contain non-function HYR1 proteins and cannot glucosylate hypostatin. Hypostatin is, therefore, a pro-drug that is activated by glucosylation, a process I have called glycoactivation. Collectively, my data show that natural variation can be used to gain insight into the mechanism of action of new compounds and that there are biochemical similarities between plant and human pharmacogenetic loci.