Proteomic and biochemical assays of glutathione-related proteins in susceptible and multiple herbicide resistant Avena fatua L.

Abstract

Extensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide resistant (MHR) Avena fatua L. populations utilized in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in U.S. small grain production, and thus pose significant agronomic and economic threats. Resistance to ALS and ACCase inhibitors is not conferred by target site mutations, indicating that non-target site resistance mechanisms are involved. To investigate the potential involvement of glutathione-related enzymes in the MHR phenotype, we used a combination of proteomic, biochemical, and immunological approaches to compare their constitutive activities in herbicide susceptible (HS1 and HS2) and MHR (MHR3 and MHR4) A. fatua plants. Proteomic analysis identified three tau and one phi glutathione S-transferases (GSTs) present at higher levels in MHR compared to HS plants, while immunoassays revealed elevated levels of lambda, phi, and tau GSTs. GST specific activity towards 1-chloro-2,4-dinitrobenzene was 1.2-fold higher in MHR4 than in HS1 plants and 1.3- and 1.2-fold higher in MHR3 than in HS1 and HS2 plants, respectively. However, GST specific activities towards fenoxaprop-P-ethyl and imazamethabenz-methyl were not different between untreated MHR and HS plants. Dehydroascorbate reductase specific activity was 1.4-fold higher in MHR than HS plants. Pretreatment with the GST inhibitor NBD-Cl did not affect MHR sensitivity to fenoxaprop-P-ethyl application, while the herbicide safener and GST inducer mefenpyr reduced the efficacy of low doses of fenoxaprop-P-ethyl on MHR4 but not MHR3 plants. Mefenpyr treatment also partially reduced the efficacy of thiencarbazone-methyl or mesosulfuron-methyl on MHR3 or MHR4 plants, respectively. Overall, the GSTs described here are not directly involved in enhanced rates of fenoxaprop-P-ethyl or imazamethabenz-methyl metabolism in MHR A. fatua. Instead, we propose that the constitutively elevated GST proteins and related enzymes in MHR plants are representative of a larger, more global suite of abiotic stress-related changes.

Stress‐induced evolution of herbicide resistance and related pleiotropic effects

“…As shown in Table , all resistant populations characterized to date exhibit various pleiotropic effects at the whole plant, biochemical, or molecular level. Of these, MHR A. fatua contains more documented molecular changes than other species, as well as several unusual resistance mechanisms, and so it has been used as a model to investigate the primary stress effects associated with the evolution of NTSR . Initial efforts to describe these have focused on constitutive differences between MHR and susceptible populations, because they are most likely to represent changes that would lead to pleiotropic effects.…”

“…92 Enhanced herbicide catabolism does not appear to play a major role in MHR A. fatua, because glutathione S-transferase specific activities towards fenoxaprop-P-ethyl and imazamethabenz-methyl were not different between untreated MHR and susceptible plants 34 and only a relatively small number of constitutively elevated xenobiotic catabolism transcripts and proteins were detected in MHR plants. 28,34 The A. fatua NTSR phenotype is heritable and stable for multiple generations in the absence of herbicide treatment. 90 Specific herbicide use histories from the fields where the MHR A. fatua populations were collected are not available, but regional practices indicate that they evolved under 40 years of chronological rotations of selective herbicides.…”

“…Of these, MHR A. fatua contains more documented molecular changes than other species, as well as several unusual resistance mechanisms, and so it has been used as a model to investigate the primary stress effects associated with the evolution of NTSR. 28,34 Initial efforts to describe these have focused on constitutive differences between MHR and susceptible populations, because they are most likely to represent changes that would lead to pleiotropic effects. Transcriptome and proteome analyses showed that MHR plants have constitutively higher levels of transcripts and proteins with functions in stress response, disease resistance, redox maintenance, xenobiotic transport and catabolism, transcriptional regulation, and signal transduction.…”

“…Although not the focus of this review, herbicide safeners also induce similar gene expression changes likely through oxylipin-mediated signaling, 32 and the unclear relationship between safeners and NTSR evolution is currently under investigation. 33,34 Given that systemic acquired acclimation and SAR are better understood than NTSR, this review proposes that the plant stress literature may be fertile ground for generating hypotheses about NTSR evolution and its pleiotropic effects.…”

You cannot fight fire with fire: a demographic model suggests alternative approaches to manage multiple herbicide‐resistant Avena fatua

“…From each generation of 50 plants, all seeds were harvested and a random selection of 50 seeds was used to initiate five additional generations without herbicide selection to homogenise the genetic background. These two populations were used to successfully characterise the biochemical mechanisms responsible for MHR (Burns et al ., ; Keith et al ., ), as well as the physiological and ecological consequences of MHR (Lehnhoff et al ., ,b). The susceptible HS1 population was derived from seeds of untreated plants bordering an adjacent barley production field, grown for seven generations as described for MHR3–4 except without herbicide selection, and was confirmed to be 100% susceptible to field rates of the herbicide used in this study (Lehnhoff et al ., ).…”