Glyphosate resistance mechanism of Arabidopsis thaliana mutant gre1; and genetic diversity in nightshade (Solanum spp.) weed species

Altanbadralt Sharkhuu, Purdue University

Abstract

Glyphosate (N-phosphonomethyl glycine) the most widely used herbicide in the world, inhibits 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), the penultimate enzyme in the shikimate pathway in plants. Resistance to glyphosate has been engineered into major agronomic crops, resulting in efficient weed control. Glyphosate resistant (GR) crops are grown on 120 million ha by 12 million farmers worldwide. The widespread use of glyphosate in GR crops has resulted in the recent appearance of 14 resistant weed biotypes. The specific mechanism(s) of resistance to glyphosate in these weeds is not known. Our research was designed to explore the possibility that glyphosate resistance mechanisms exist other than altered function of EPSPS or glyphosate catabolism. Using forward genetic screening with T-DNA tagged Arabidopsis thaliana (Col-0) populations we identified and characterized several glyphosate response mutants. One of these mutants is named gre1-1 ( glyphosate response 1-1). Mutation of gre1-1 caused dysfunction of phyB, the red and far red light receptor. Gre1 has elevated resistance to in vitro glyphosate treatment but a slightly lower EPSPS expression compared to wild type (WT). However, in vitro enzyme activity assays showed gre1-1 EPSPS was as sensitive to glyphosate as WT. The gre1-1 mutant also has lower levels of shikimate accumulation than WT, consistent with its increased resistance. Glyphosate uptake, translocation and shikimate pathway activity studies showed that gre1-1 absorbed more but translocated less glyphosate to the growing region than WT. Gre1-1 recovered after glyphosate spray treatment while WT died. This resurrecting phenotype could be an indication of hypersensitive (HR) response mediated by salicylic acid (SA). One of downstream pathways initiated from the shikimate pathway produces salicylic acid (SA). SA pretreatment caused a suppression of the glyphosate resistant phenotype of gre1-1 indicating induction of HR. It was also observed that glyphosate treatment resulted in lower expression of the plastidial membrane phosphate transporter PHT2;1 and in lower accumulation of anthocyanin in gre1-1, both common responses to stress. These results indicate that light signaling is an important part of plant responses to glyphosate and, indeed the glyphosate resistance of gre1-1 in response to light is altered. The genetic diversity of solanaceous weed species including eastern black nightshade, black nightshade, American black nightshade, bitter nightshade, clammy groundcherry, smooth groundcherry and horsenettle was determined by Random Amplification of Polymorphic DNA (RAPD). The unweighted pair group method with arithmetic average (UPGMA) analysis allowed us to divide these species into 11 clusters. At > 0.85 similarity coefficient eastern black nightshade, black nightshade and American black nightshade were more genetically similar to each other than to other species while bitter nightshade and ground cherry were distinct. All but three of the eastern black nightshade accessions were in the same cluster. Accessions from species other than eastern black nightshade separated into distinct clusters for one accession of black nightshade that RAPD's identified as American black nightshade. Results confirm RAPD analysis of genetic polymorphisms is useful to characterize weedy solanaceous species.

Degree

Ph.D.

Advisors

Weller, Purdue University.

Subject Area

Molecular biology|Genetics|Plant biology

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