Metabolism of the 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor, Mesotrione, in Multiple-Herbicide-Resistant Palmer amaranth (Amaranthus palmeri)

Abstract

Metabolic resistance to the maize-selective, HPPD-inhibiting herbicide, mesotrione, occurs via Phase I ring hydroxylation in resistant waterhemp and Palmer amaranth; however, mesotrione detoxification pathways post-Phase I are unknown. This research aims to (1) evaluate Palmer amaranth populations for mesotrione resistance via survivorship, foliar injury, and aboveground biomass, (2) determine mesotrione metabolism rates in Palmer amaranth populations during a time course, and (3) identify mesotrione metabolites including and beyond Phase I oxidation. The Palmer amaranth populations, SYNR1 and SYNR2, exhibited higher survival rates (100%), aboveground biomass (c.a. 50%), and lower injury (25-30%) following mesotrione treatment than other populations studied. These two populations also metabolized mesotrione 2-fold faster than sensitive populations, PPI1 and PPI2, and rapidly formed 4-OH-mesotrione. Additionally, SYNR1 and SYNR2 formed 5-OH-mesotrione, which is not produced in high abundance in waterhemp or naturally tolerant maize. Metabolite features derived from 4/5-OH-mesotrione and potential Phase II mesotrione-conjugates were detected and characterized by liquid chromatography-mass spectrometry (LCMS).

Keywords: cytochrome P450 monooxygenase; dioecious amaranth; herbicide detoxification; mesotrione resistance; metabolomics; oxidative metabolism; waterhemp.

Figure 1

Structure of the 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide, mesotrione, [2-(4-(methylsulfonyl)-2-nitrobenzoyl)cyclohexane-1,3-dione].

Figure 2

Visual assessments of plant injury (A) and aboveground dry biomass (B) at 14 DAT of 105 g a.i. ha^–1 mesotrione plus 1% (v/v) COC and 2.5% (v/v) AMS in eight Palmer amaranth (Amaranthus palmeri) and two waterhemp (A. tuberculatus) populations. Error bars correspond to the standard error of the mean for plant injury and dry biomass across 20 replicates. The difference in letters corresponding to each population denotes significant differences at p ≤ 0.05. p-value adjustment was performed using Tukey’s method for comparing a family of 10 estimates.

Figure 3

Representative plants of Palmer amaranth (Amaranthus palmeri) populations: PS-14617 (A), PS-9857 (B), PS-8398 (C), and PS-11907 (D) after treatment with 105 g a.i. ha^–1 mesotrione plus 1% (v/v) COC and 2.5% (v/v) AMS at 14 DAT and compared with their respective untreated controls (adjuvants only). These populations are henceforth called SYNR1, SYNR2, PPI1, and PPI2, respectively.

Figure 4

Silica gel thin-layer chromatography of mesotrione metabolites in four Palmer amaranth (Amaranthus palmeri) and two waterhemp (A. tuberculatus) (underlined) at 2 and 12 h after treatment (HAT) with [¹⁴C]-mesotrione, as well as maize at 2, 8, and 24 HAT. Each lane corresponds to a sample: (1, 18) Origin of spotting and solvent front; (2) SIR, 2 h; (3) SIR, 12 h; (4) SYNR1, 2 h; (5) SYNR1, 12 h; (6) SYNR2, 2 h; (7) SYNR2, 12 h; (8) ACR, 2 h; (9) ACR, 12 h; (10) PPI1, 2 h; (11) PPI1, 12 h; (12) PPI2, 2 h; (13) PPI2, 12 h; (14) Maize, 2 h; (15) Maize, 8 h; (16) Maize, 24 h; and (17) [¹⁴C]-mesotrione standard in 0.1 M Tris-Cl (pH 6.0). Maize extracts were obtained from a separate assay and used as a positive control for in vivo production of the 4-hydroxy-mesotrione metabolite (M2, blue box). M1, putative 5-hydroxy-mesotrione metabolite (pink box).

Figure 5

Metabolite profiling of mesotrione-resistant Palmer amaranth (Amaranthus palmeri) (SYNR1 and SYNR2), multiple-herbicide-resistant waterhemp (A. tuberculatus) (SIR), and mesotrione-sensitive Palmer amaranth (PPI1) and waterhemp (ACR) populations. (A) Principal components analysis scores scatter plot comparing Palmer amaranth and waterhemp (PC1 = 23.6%; PC2 = 15.1%). (B) PLS-DA scores plot of five populations at Component 1 (19.6%) and mesotrione resistance on Component 2 (15.7%). (C) Percentage of mesotrione at 2 HAT remaining in excised leaf extracts at 6 and 12 HAT. Differences in letters corresponding to each population per HAT denote a significant difference at p ≤ 0.05. p-value adjustment was performed using Tukey’s method for comparing a family of 10 estimates. (D) Relative percent abundance of 4- to 5-hydroxy-mesotrione metabolites (4-OH to 5-OH) at 12 HAT. Asterisks indicate significant differences compared to SIR via analysis of variance followed by Tukey’s multiple comparisons of means at α = 0.05. Error bars correspond to the standard error of the mean across four replicates.

Figure 6

Mass spectra and chemical structures of mesotrione (A), 4-OH mesotrione (B), and 5-OH mesotrione (C) in Palmer amaranth (Amaranthus palmeri) and waterhemp (A. tuberculatus). Identities of these compounds were confirmed using analytical standards.

Figure 7

Metabolism of mesotrione in four Palmer amaranth (Amaranthus palmeri) populations (PPI1, PPI2, SYNR1, and SYNR2). (A) Principal components analysis scores scatter plot of four Palmer amaranth populations with the four biological replicates at five time points (2, 4, 8, 12, and 24 h after treatment (HAT)). (B) Loading scatter plot of metabolite features from these populations detected via liquid chromatography–mass spectrometry. (C) Degradation of mesotrione (parent) herbicide throughout a time-course study. (D) Heatmap dendrogram comparing the 25 most discriminating metabolite features in mesotrione-treated Palmer amaranth populations and untreated control (Control) samples at 24 HAT.

Figure 8

Correlation plot based on normalized relative peak abundance of mesotrione and its identified and putative metabolites detected via liquid chromatography–mass spectrometry of Amaranthus palmeri leaf extracts treated with 0.15 mM nonlabeled mesotrione across five time points (2, 4, 8, 12, and 24 h after treatment). The strength and direction of the correlation are represented by Pearson’s r values indicated by each box color based on the scale of −1.0 (violet) to +1.0 (red).

Figure 9

Proposed metabolic detoxification pathway for mesotrione in multiple-herbicide-resistant (MHR) Amaranthus palmeri leaves. Phase I hydroxylated metabolites of mesotrione were verified by liquid chromatography–mass spectrometry (−OH groups are circled in green). Potential Phase II conjugates of mesotrione (sulfate and sugar groups are circled in pink) are theoretical metabolites based on their mass spectral data.