Abstract
Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15's substrate preference. The X-ray crystal structure of the AtGH3.15·2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-catalyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.
Highlights
This suggests that 2,4-D is a potent IAA analog targeting the TIR1 auxin receptor, it does not serve as an IAA mimic for the GH3 proteins
The Km values reported for various GH3 proteins with their cognate plant hormone substrates are typically in the 300 – 800 M range; overexpression and knockout plant lines of the different GH3-encoding genes exhibit growth phenotypes with phytohormone treatments in the range of 1 to 10 M that correspond to GH3 protein expression changes [25, 26, 30, 32, 33]. These differences highlight the need for further investigations into the metabolism of these molecules, which may alter local concentrations within different tissues and cell types of the plant and the fluxes that control plant growth and development
This parallels the effect of treating Arabidopsis AtGH3.15 knockout and overexpression lines with the auxin IBA [33]
Summary
Screen of Arabidopsis GH3 proteins with 2,4-D and 2,4-DB and comparison to auxin substrates. Steady-state kinetics with cysteine, histidine, methionine, glutamine, and tyrosine were determined and confirm that, as with IBA, glutamine is the preferred amino acid for AtGH3.15 with 2,4-DB (Table 2). Comparison with the position of AMP in the AtGH3.151⁄7AMP complex indicates that only one 2,4-DB molecule is positioned in an orientation that points the reactive carboxylate group toward the phosphate group that undergoes the adenylation reaction (Fig. 3C) This 2,4-DB molecule stacks with Phe-166, forms a hydrogen bond contact with Ser-122 (which was modeled in two alternate side-chain conformations), and is situated in a space bordered by Met-162, Val-163, Phe-325, and Phe-332 (Fig. 3D). The substituted phenyl ring is positioned to form van der Waals contacts with Ile-143, Leu-181, and Phe219 It is not clear if the binding of two 2,4-DB molecules in the acyl acid site of AtGH3.15 is biochemically relevant or is an artifact of crystallization. M 530 Ϯ 40 3790 Ϯ 420 590 Ϯ 100 23,200 Ϯ 2300 1130 Ϯ 100 3550 Ϯ 520 180 Ϯ 70 14,800 Ϯ 2470 2140 Ϯ 260 6000 Ϯ 420 960 Ϯ 50 680 Ϯ 50 kcat/Km
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