With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.
Increasing the pH of the spray water to solubilize the weak acid herbicide nicosulfuron and then decreasing pH below its pKaso that it converts into a neutral form enhances biological activity under some conditions. The water-dispersible granule formulation of nicosulfuron starts as dispersed particles. Adding 1% wt/wt K3PO4solubilizes nicosulfuron and increases its activity compared to its dispersion without base. The type of buffer and the surfactant HLB or hydrophilic lipophilic balance, a measure of the molecular balance of the hydrophilic and lipophilic groups, altered the activity of nicosulfuron. Adding 1% wt/wt K3PO4increases the pH, and the optimum HLB ranged from 13 to 17 on large crabgrass. Adding 1% wt/wt H3PO4reduces the pH and lowers the optimum HLB range from 10 to 14 on large crabgrass. Adding the acidic buffer converts the solubilized nicosulfuron into its neutral form and increases activity under some surfactant conditions. Thus, neutral nicosulfuron is more active with lipophilic surfactants, while ionic nicosulfuron is more active with hydrophilic surfactants. When tested on other species, low HLB surfactants are the most active at low pH. These results support the concept that the physicochemical properties of the herbicide, adjuvants, and weed species should be matched for optimum activity.
The transgenic corn line 98140 has a high level of resistance to glyphosate and all five chemical classes of herbicides that inhibit acetolactate synthase (ALS). The dual herbicide resistance is due to a molecular stack of two constitutively expressed genes: gat4621, which produces a glyphosate acetyltransferase that rapidly inactivates glyphosate, and hra, which produces a highly resistant ALS. On a rate basis, the positive 98140 isoline with a single copy of the gat4621 gene is over 1,000-fold more resistant to glyphosate than a negative isoline without the transgene. Similarly, the positive 98140 isoline with the hra gene is over 1,000-fold more resistant to ALS-inhibiting herbicides such as chlorimuron and sulfometuron at the whole-plant and enzyme level. The gat4621 and hra genes do not change the natural tolerance of corn to selective herbicides, so new corn hybrids based on 98140 will give growers more options to manage weeds and delay the evolution of herbicide-resistant weeds.
The pH of the spray mixture controls the solubility and ionic state of weak acid herbicides and thus influences their uptake and biological activity. When the pH of the spray water is below the pKa of the herbicide, increasing pH can increase solubility and improve activity when herbicide solubility limits uptake. However, raising the pH above the pKa makes the weak acid anionic and thus may make it more difficult to penetrate into the lipophilic cuticle and the negatively charged membrane and cell wall. Decreasing the pH below the pKa converts the weak acid into a neutral or unionized form and thus makes it easier to penetrate these lipophilic and negatively charged barriers. pH also influences other herbicide properties including chemical stability, volatility, and chemical compatibility. Thus, manufacturers need to balance a number of properties when they adjust the pH of their adjuvant and herbicide formulations. These studies show significant differences in the biological activity of several herbicides when the pH is increased and decreased with a range of surfactant types. These results support the concept that the physicochemical properties of the herbicide and adjuvant should be matched for optimum activity.
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