Metabolic Pathway of Topramezone in Multiple-Resistant Waterhemp (Amaranthus tuberculatus) Differs From Naturally Tolerant Maize

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a problematic dicot weed in maize, soybean, and cotton production in the United States. Waterhemp has evolved resistance to several commercial herbicides that inhibit the 4-hydroxyphenylpyruvate-dioxygenase (HPPD) enzyme in sensitive dicots, and research to date has shown that HPPD-inhibitor resistance is conferred by rapid oxidative metabolism of the parent compound in resistant populations. Mesotrione and tembotrione (both triketones) have been used exclusively to study HPPD-inhibitor resistance mechanisms in waterhemp and a related species, A. palmeri (S. Wats.), but the commercial HPPD inhibitor topramezone (a pyrazolone) has not been investigated from a mechanistic standpoint despite numerous reports of cross-resistance in the field and greenhouse. The first objective of our research was to determine if two multiple herbicide-resistant (MHR) waterhemp populations (named NEB and SIR) metabolize topramezone more rapidly than two HPPD inhibitor-sensitive waterhemp populations (named SEN and ACR). Our second objective was to determine if initial topramezone metabolite(s) detected in MHR waterhemp are qualitatively different than those formed in maize. An excised leaf assay and whole-plant study investigated initial rates of topramezone metabolism (<24 h) and identified topramezone metabolites at 48 hours after treatment (HAT), respectively, in the four waterhemp populations and maize. Results indicated both MHR waterhemp populations metabolized more topramezone than the sensitive (SEN) population at 6 HAT, while only the SIR population metabolized more topramezone than SEN at 24 HAT. Maize metabolized more topramezone than any waterhemp population at each time point examined. LC-MS analysis of topramezone metabolites at 48 HAT showed maize primarily formed desmethyl and benzoic acid metabolites, as expected based on published reports, whereas SIR formed two putative hydroxylated metabolites. Subsequent LC-MS/MS analyses identified both hydroxytopramezone metabolites in SIR as different hydroxylation products of the isoxazole ring, which were also present in maize 48 HAT but at very low levels. These results indicate that SIR initially metabolizes and detoxifies topramezone in a different manner than tolerant maize

Cytochrome P450 Inhibitors Reduce Creeping Bentgrass (Agrostis stolonifera) Tolerance to Topramezone

Creeping bentgrass (Agrostis stolonifera L.) is moderately tolerant to the p-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide topramezone. However, the contribution of plant metabolism of topramezone to this tolerance is unknown. Experiments were conducted to determine if known cytochrome P450 monooxygenase inhibitors 1-aminobenzotriazole (ABT) and malathion alone or in combination with the herbicide safener cloquintocet-mexyl influence creeping bentgrass tolerance to topramezone. Creeping bentgrass in hydroponic culture was treated with ABT (70 μM), malathion (70 μm and 1000 g ha(-1)), or cloquintocet-mexyl (70 μM and 1000 g ha(-1)) prior to topramezone (8 g ha(-1)) application. Topramezone-induced injury to creeping bentgrass increased from 22% when applied alone to 79 and 41% when applied with malathion or ABT, respectively. Cloquintocet-mexyl (70 μM and 1000 g ha(-1)) reduced topramezone injury to 1% and increased creeping bentgrass biomass and PSII quantum yield. Cloquintocet-mexyl mitigated the synergistic effects of ABT more than those of malathion. The effects of malathion on topramezone injury were supported by creeping bentgrass biomass responses. Responses to ABT and malathion suggest that creeping bentgrass tolerance to topramezone is influenced by cytochrome P450-catalyzed metabolism. Future research should elucidate primary topramezone metabolites and determine the contribution of cytochrome P450 monooxygenases and glutathione S-transferases to metabolite formation in safened and non-safened creeping bentgrass

Transcriptome Profiling and Genome-Wide Association Studies Reveal GSTs and Other Defense Genes Involved in Multiple Signaling Pathways Induced by Herbicide Safener in Grain Sorghum

Herbicide safeners protect cereal crops from herbicide injury by inducing genes and proteins involved in detoxification reactions, such as glutathione S-transferases (GSTs) and cytochrome P450s (P450s). Only a few studies have characterized gene or protein expression profiles for investigating plant responses to safener treatment in cereal crops, and most transcriptome analyses in response to safener treatments have been conducted in dicot model species that are not protected by safener from herbicide injury. In this study, three different approaches were utilized in grain sorghum (Sorghum bicolor (L.) Moench) to investigate mechanisms involved in safener-regulated signaling pathways. An initial transcriptome analysis was performed to examine global gene expression in etiolated shoot tissues of hybrid grain sorghum following treatment with the sorghum safener, fluxofenim. Most upregulated transcripts encoded detoxification enzymes, including P450s, GSTs, and UDP-dependent glucosyltransferases (UGTs). Interestingly, several of these upregulated transcripts are similar to genes involved with the biosynthesis and recycling/catabolism of dhurrin, an important chemical defense compound, in these seedling tissues. Secondly, 761 diverse sorghum inbred lines were evaluated in a genome-wide association study (GWAS) to determine key molecular-genetic factors governing safener-mediated signaling mechanisms and/or herbicide detoxification. GWAS revealed a significant single nucleotide polymorphism (SNP) associated with safener-induced response on chromosome 9, located within a phi-class SbGST gene and about 15-kb from a different phi-class SbGST. Lastly, the expression of these two candidate SbGSTs was quantified in etiolated shoot tissues of sorghum inbred BTx623 in response to fluxofenim treatment. SbGSTF1 and SbGSTF2 transcripts increased within 12-hr after fluxofenim treatment but the level of safener-induced expression differed between the two genes. In addition to identifying specific GSTs potentially involved in the safener-mediated detoxification pathway, this research elucidates a new direction for studying both constitutive and inducible mechanisms for chemical defense in cereal crop seedlings