|Plaisance, Kathryn - UNIVERSITY OF MINNESOTA|
Submitted to: Pesticide Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 20, 1998
Publication Date: N/A
Interpretive Summary: Herbicide-resistant weeds have sometimes appeared in fields where the same herbicide has been applied for a number of years. Atrazine is a herbicide that normally provides good control of velvetleaf, a weed commonly found in cornfields. However, several years ago a velvetleaf biotype that was not controlled by atrazine was found in a farmer's field in Maryland. The reason why this weed was resistant to atrazine was not known. Our previous work indicated that resistance was due to the ability of the weed to rapidly deactivate atrazine by linking it with glutathione, a naturally occurring compound found in plant cells. This reaction was catalyzed by plant proteins known as glutathione S-transferases (GSTs). These proteins not only detoxify foreign compounds such as herbicides, but also play a role in protecting plants from various environmental stresses. We developed a protocol to isolate GST proteins from atrazine-resistant and atrazine-susceptible velvetleaf weeds and compared their properties. We found that the GSTs in the resistant biotype differed from those in the susceptible biotype because they were able to detoxify atrazine much faster which resulted in herbicide resistance. Knowledge generated by this research can be used to develop strategies to prevent the occurrence and reduce the spread of herbicide-resistant weeds. In addition, the protocol we developed for isolating GSTs from plant cells will be useful in future research to learn more about how these proteins protect plants from other types of environmental stress.
Technical Abstract: Glutathione S-transferases (GST, EC 126.96.36.199) were purified to homogeneity from leaves of atrazine-resistant and -susceptible velvetleaf (Abutilon theophrasti, Medic.) biotypes using a protocol involving DEAE-anion exchange, S-hexylglutathione affinity, and Superose 12-gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity-purified GST dimers from both resistant and susceptible biotypes indicated the presence of three GST subunits with Mr of 27,000, 26,000, and 25,000. GST subunits in both resistant and susceptible biotyes were glycosylated as revealed by probing western blots with concanavalin A/avidin-alkaline phosphatase. Isoelectric focusing indicated two major GST isoforms in both biotypes with pI values of 4.4 and 4.7. Initial-rate kinetic analysis of GST(atrazine) activity in the affinity-purified dimeric GST fraction from both resistant and susceptible biotypes indicated a sequential, rapid-equilibrium, random Bi-Bi kinetic mechanism. Kinetic analysis of GST(atrazine) activity from resistant and susceptible biotypes indicated no significant difference in Km values for GSH and atrazine. However, the catalytic constant (Kcat) was approximately three-fold greater for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Inhibition constants (Ki values) for the GSH-atrazine conjugate were approximately 1.5-fold higher for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Enhanced Kcat for GST(atrazine) activity measured in the purified GST fraction from the atrazine-resistant biotype is due, in part, to reduced end-product inhibition by the glutathione-atrazine conjugate.