4.1. Plant Material and Growing Conditions
Four wheat genotypes were used to compare whole-plant resistance of lines with wildtype and mutant ACCase as well as specific activity and resistance of extracted ACCase. The AF10 and Hatcher lines were provided by Dr. Scott Haley from the Department of Crop and Soil Science, and the varieties Byrd, and Incline AX were obtained from the Colorado Wheat Research Foundation. All experiments carried out on these wheat varieties were done with their permission and complied with local and national regulations. Byrd is a quizalofop-susceptible variety that was the recurrent parent for Incline AX, a CoAXium® variety, whereas Hatcher is the original mutagenized variety. AF10 is one of the initial quizalofop-resistant lines, which contains a homozygous mutant ACC1 homoeolog in sub-genome D10. Incline AX exhibits two mutant homoeologs in sub-genomes A and D. Byrd was the wildtype, null mutant in all physical experiments except for the quizalofop plant growth dose-response experiment, where Hatcher was substituted. AF10 and Incline AX were consistently used as single and double mutants, respectively, in the same experiments.
Plants for the whole-plant dose responses and ACCase extractions were grown from seed in soilless media. Plants were maintained in a greenhouse environment with temperatures from 20 to 25°C, relative humidity between 50 to 70%, and a 14-hour daylength with natural lighting supplemented by sodium halide lamps.
4.2. Herbicide Dose Effect on Plant Growth
Prior to reaching the jointing stage, wheat seedlings were treated with herbicide. Untreated seedlings of each line were also retained to normalize data. Eleven rates of herbicide were used in each dose-response experiment. Quizalofop herbicide doses for the susceptible line ranged from 3.85 and 108 g ae ha-1, whereas doses for resistant lines ranged from 15.4 and 493 g ae ha-1. Haloxyfop doses ranged from 15.4 and 616 g ae ha-1 for all wheat lines. Non-ionic surfactant was added to each dose (0.25% v/v). All treatments were applied in a single pass at 40 cm above the plant canopy using a spray chamber. Each treatment was triplicated, with four plants comprising a replicate. Fresh leaf biomass was harvested three weeks after treatment. Biomass was adjusted in some instances to account for germination of less than four plants per replicate.
4.3. Enzyme Extraction
Approximately 5.0 g of crown tissue was harvested from each genotype at the two-leaf stage, flash frozen in liquid nitrogen, and stored at -80°C. Enzyme extraction and activity assay were modified from previously published methods12–14. Frozen wheat tissue was powdered in liquid nitrogen. All subsequent extraction steps were carried out on ice or in a cold room to maintain an extract temperature of 4°C. Ground tissue was transferred to 14.85 mL extraction buffer (100 mM Trizma, 20 mM DTT, 2 mM l-ascorbic acid, 1 mM EDTA, 0.5% w/v PVP-40, 0.5% w/v insoluble PVP-20, and 10% v/v glycerol; pH 8.0) and 150 µL of 100 mM PMSF (dissolved in 100% ethanol). The tissue was homogenized at max speed for 30 s with a homogenizer probe. Homogenate was filtered by hand with Miracloth, followed by centrifugation for 30 min at a speed of 25,000 x g. The resulting supernatant was slowly brought to 66% saturation with solid ammonium sulfate and stirred for an hour.
Ammonium sulfate solutions were centrifuged using the same parameters as the previous centrifugation step to precipitate ACCase and similar proteins. After discarding the supernatant, the protein pellets were resuspended with 2.5 mL elution buffer (50 mM tricine, 50 mM KCl, 2.5 mM MgCl2, and 1 mM DTT; pH = 8.0). The protein extracts were desalted using a gravity protocol with conditioned PD-10 columns. Desalted protein extracts were eluted with 3.5 mL of elution buffer and mixed with 25% v/v glycerol.
4.4. Enzyme Specific Activity Assay
To determine ACCase specific activity, four 40 µL aliquots of fresh protein extract per genotype were preincubated for 3 min in assay solution without acetyl-CoA. Acetyl-CoA was subsequently added to three aliquots to initiate enzyme activity, whereas water was added in lieu of acetyl-CoA for a background readings. Preincubation and reaction steps were maintained at 32°C with moderate shaking. Final concentrations of assay solution components for each 200 µL reaction (including enzyme aliquot) were as follows: 20 mM tricine (pH = 8.3), 10 mM KCl, 10 mM MgCl2, 5 mM ATP, 3.24 mM NaHCO3, 2.5 mM DTT, 0.1% w/v BSA, 0.25 mM acetyl-CoA, and 0.25 mM NaH14CO3 (provides 18.5 kBq per reaction). After 10 min, enzyme reactions were quenched with 20 µL of 12 M hydrochloric acid. Solutions were transferred to 20 mL scintillation vials and left uncapped overnight to enable volatilization of unincorporated carbonate to CO2. Reactions were carried out in a fume hood equipped with a 14C filter.
The following day, 10 mL of scintillation cocktail was added to each vial. Vials were capped and vortexed for 20 s prior to analysis. Radioactivity was measured with a liquid scintillation analyzer in disintegrations per min (DPM).
4.5. Herbicide Dose Effect on ACCase Activity
Specific activity dose-responses were conducted using the same protocol as the specific activity assay but with the addition of herbicide immediately preceding the preincubation step. Dilution series were prepared with quizalofop acid and haloxyfop acid analytical standards dissolved in 100% acetonitrile. Triplicated dose treatments per genotype included background readings (water added in lieu of acetyl-CoA), null herbicide doses, and doses that provided 0.0100, 0.100, 1.00, 10.0, 100, and 1000 µM herbicide in the final assay solution.
4.6. Homology Modeling of Wheat Carboxyl Transferase Domain
The segments of wheat ACCase sequence where the A2004V substitution is located were aligned to the sequence of the CT domain of yeast ACCase using EMBOSS Needle15. This information was used to build a homology model of wheat ACCase based on the crystal coordinates of the carboxyl transferase (CT) domain of yeast ACCase (1UYS)16 available from the RCSB protein data bank. The homology modeling pipeline was similar to that reported previously to model the binding of glufosinate on resistant glutamine synthetase from Lolium perenne17. A preliminary model was obtained using Modeller 10.018,19. The model was assembled in its functional dimer configuration and refined using GROMACS (version 2018.3)20,21 on a workstation with two processors (96 threads) and video cards (10 GB GPU memory each). Steric clashes or inappropriate geometries were corrected through molecular dynamics simulation and evaluated using MolProbity22,23 as described before.
Proteins and ligand interactions were visualized using PyMOL v.2.3.324. Additionally, the docking of quizalofop and haloxyfop to native and mutant carboxyl transferase dimers of wheat was measured using established protocols developed for Autodock25,26. Briefly, the receptor was defined to encompass residues lining the herbicide binding domain. Amino acid charges were calculated using Gasteiger charges including all polar hydrogens27. The docking imposed a covalent map to involve the nitrogen hydrogen of ILE 222 in the docking of the herbicides within the gridbox. A total of 100 poses were generated.
4.7. Statistical Analysis
All statistical analysis was conducted with packages in RStudio v.1.2.5033 with R "Orange Blossom”28. To model the effect of herbicide on plant growth, fresh weight data was first normalized to percent of control means. Data was transformed with a Box-Cox transformation. For quizalofop data, a two-parameter log-logistic regression was fit using the LL.4 [drc] function:
$$y=c-\frac{d-c}{1+\text{e}\text{x}\text{p}\left(m\left[\text{log}\left(x\right)-\tilde{e}\right]\right)}$$
1
,
where lower limit c is fixed at 0, upper limit d is fixed at 100, m is the slope, and e is the 50% growth reducing dose (GR50). A four-parameter Brain-Cousens model was fit using the BC.4 [drc] function for haloxyfop data:
$$y=c+\frac{d-c+f}{1+\text{e}\text{x}\text{p}\left(m\left[\text{log}\left(x\right)-\tilde{e}\right]\right)}$$
2
,
where lower limit c is fixed at 0, d is the upper limit, f is the hormesis effect, with m and e as additional parameters without direct interpretation. The GR50 of the haloxyfop model was estimated through parameterization with the ED [drc] function. A resistance factor (R:S50) was calculated by dividing GR50’s of quizalofop-resistant wheat lines by the GR50 of a wildtype, susceptible line with the EDcomp [drc] function. Student’s t-tests (a = 0.05) of resistance factor ratios were used to identify significant fold differences in resistance between estimated GR50 doses.
A linear calibration curve of absorbance versus BSA concentration was fit using the stats [lm] function to estimate wheat ACCase extract protein concentrations:29
,
where m and b constants and standard deviations were 2.76·10− 3 ± 3.21·10− 4 and 1.40·10− 1 ± 1.32·10− 3, respectively. The residual standard error of the calibration was 1.06·10− 3 with a multiple R-squared value of 0.987. Genotype protein concentrations were calculated from the calibration and adjusted for dilution.
Per genotype, the DPM measurement of a reaction to which water was added instead of acetyl-CoA was subtracted from replicate specific activity measurements as background activity. Mean DPM enzyme specific activity measurements were converted to 14C pmol units using manufacturer provided activity of 14C-labeled sodium bicarbonate. Specific activity was then weighted by protein concentration to yield units of 14C pmol·mg− 1 protein. Standard errors reflect error propagation of DPM measurement replicates and protein concentration. Specific activity was compared between genotypes using ordinary one-way ANOVA F-protected Student’s t-tests (a = 0.05, n = 3) with anova [stats] and emmeans [emmeans] functions.
For specific activity dose-response analysis, background activity was subtracted from DPM readings. Background corrected DPM readings were normalized to percent residual activity using control means for each genotype. Data was modelled using the same methods as for the effect of quizalofop on whole-plant growth, with the exception that e represents the 50% enzyme inhibition dose (I50). Similar resistance factors (R:S) were calculated using I50 estimates, followed by Student’s t-tests (a = 0.05) of ratios to identify significant fold differences in resistance between estimated doses.