Claire A. CaJacob

Investigations into glyphosate-resistant horseweed (Conyza canadensis): retention, uptake, translocation, and metabolism

Abstract The mechanism of glyphosate resistance in horseweed was investigated. Eleven biotypes of putative sensitive (S) and resistant (R) horseweed were obtained from regions across the United States and examined for foliar retention, absorption, translocation, and metabolism of glyphosate. Initial studies used spray application of 14C-glyphosate to simulate field application. When S and R biotypes were compared in the absence of toxicity at a sublethal dose, we observed comparable retention and absorption but reduced root translocation in the R biotypes. S and R biotypes from Delaware were further examined at field use rates and results confirmed similar retention and absorption but reduced root translocation in the R biotypes. Application of 14C-glyphosate to a single leaf demonstrated reduced export out of the treated leaf and lower glyphosate import into other leaves, the roots, and the crown in R relative to S biotypes. Examination of the treated leaf by autoradiography showed that glyphosate loading into the apoplast and phloem was delayed and reduced in the R biotype. Our results consistently showed a strong correlation between impaired glyphosate translocation and resistance. Tissues from both S and R biotypes showed elevated levels of shikimate suggesting that 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) remained sensitive to glyphosate. Analysis of tissue shikimate levels demonstrated reduced efficiency in EPSPS inhibition in the R biotypes. Our results suggest that resistance is likely due to altered cellular distribution that impaired phloem loading and plastidic import of glyphosate resulting in reduced overall translocation as well as inhibition of EPSPS. Nomenclature: Glyphosate; horseweed, Conyza canadensis (L.) Cronq. ERICA.

Discovery of imidazole glycerol phosphate dehydratase inhibitors through 3-D database searching

Imidazole glycerol phosphate dehydratase (IGPD) has become an attractive target for herbicide discovery since it is present in plants and not in mammals. Currently no knowledge is available on the 3-D structure of the IGPD active site. Therefore, we used a pharmacophore model based on known inhibitors and 3-D database searches to identify new active compounds. In vitro testing of compounds from the database searches led to the identification of a class of pyrrole aldehydes as novel inhibitors of IGPD.

Design and synthesis of β-carboxamido phosphonates as potent inhibitors of imidazole glycerol phosphate dehydratase

We describe the synthesis and enzymatic activity of a library of beta-carboxamido phosphonates as inhibitors of imidazole glycerol phosphate dehydratase (IGPD). Biological results suggest the presence of an enzymatic interaction site not previously observed for other inhibitors of IGPD.

Suppression of CtpA in Mouseearcress Produces a Phytotoxic Effect: Validation of CtpA as a Target for Herbicide Development

Abstract To validate carboxyterminal processing protease of D1 protein (CtpA) as a target for herbicide discovery, CtpA sense mRNAs were overexpressed to suppress the internal level of CtpA protein in mouseearcress plants. Using antibodies raised against recombinant CtpA protein, we demonstrated that we have generated transgenic mouseearcress plants with reduced levels of CtpA protein and plants with elevated levels of CtpA protein. Transgenic plants with severely reduced levels of CtpA protein exhibited a bleached and chlorotic phenotype and stunted growth. The mutant phenotypes were enhanced by bright illumination. However, plants with a slight reduction of CtpA protein did not exhibit the mutant phenotype and could not be distinguished from wild-type plants under normal growth conditions. Several CtpA enzyme inhibitors were shown to have herbicidal activity in planta. Interestingly, plants producing excessive amount of CtpA protein were shown to be resistant to these inhibitors. Our results suggest that CtpA is essential for plant growth and development, but a reduced amount of CtpA is sufficient to carry out its essential function. CtpA may be a good target for herbicide development, but very high levels of inhibition may be required to produce a herbicidal effect. In addition, overexpressing CtpA in target plants might provide a mechanism for producing plants resistant to the herbicide. Nomenclature: CtpA, Carboxyterminal processing protease of D1 protein (also referred to as D1 protease); mouseearcress, Arabidopsis thaliana (L.) Heynh

Discovery, Development, and Commercialization of Roundup Ready® Crops

The herbicide Roundup® whose active ingredient is glyphosate (N-[phosphonomethy1]-glycine) was introduced as a broad spectrum, post emergent herbicide in 1974. Extensive research by a number of groups showed that glyphosate’s herbicidal activity resulted from inhibition of the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) a key enzyme in the shikimate pathway that is responsible for the biosynthesis of aromatic amino acids in plants and microbes (reviewed, Franz et al., 1997).